WO2020053196A1 - Method for the biocatalytic alkylation of a substrate - Google Patents

Method for the biocatalytic alkylation of a substrate Download PDF

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Publication number
WO2020053196A1
WO2020053196A1 PCT/EP2019/074076 EP2019074076W WO2020053196A1 WO 2020053196 A1 WO2020053196 A1 WO 2020053196A1 EP 2019074076 W EP2019074076 W EP 2019074076W WO 2020053196 A1 WO2020053196 A1 WO 2020053196A1
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Prior art keywords
group
methyl
methyltransferase
carrier compound
sulfur
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PCT/EP2019/074076
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French (fr)
Inventor
Florian SEEBECK
Cangsong LIAO
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Universität Basel
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes

Definitions

  • the present invention relates to a method for alkylating a substrate comprising an alkylation step a) wherein a S-alkyltransferase transfers an alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated carrier compound and an alkylation step b) wherein a N-, C-, O-, S- or P- alkyitransferase transfers the alkyl group from the alkylated carrier compound to the substrate, yielding an alkylated substrate and a dealkylated carrier compound, and wherein at least a part of the dealkylated carrier compound is recycled to regenerate the alkylated carrier compound.
  • the invention relates to a method for producing the alkylated sulfur- or selenium-containing carrier compound.
  • the invention also relates to kits for alkylating a substrate or for producing the alkylated carrier compound and the use of the components and kits for the production of an alkylated substrate or an alkylated carrier compound.
  • Alkylation in particular methylation
  • alkylation is a common reaction in natural product biosynthesis and in signal transduction.
  • Introduction of alkyl groups, in particular methyl groups, onto small and large biomolecules can change their physiochemical properties significantly. Therefore, alkylation, in particular methylation, is an important synthetic approach to optimize the potency and pharmacokinetic properties of therapeutic compounds.
  • alkyl transfers are conceptionally simple, attachment of alkyl groups to specific functions on complex molecules is challenging. The development of synthetic methodologies to overcome this problem is an important frontier in organic chemistry.
  • SAM S-adenosyl methionine
  • the technical problem underlying the present invention is thus the provision of a system for improved enzyme-catalyzed alkylation.
  • step b) wherein at least a part of the dealkyiated sulfur- or selenium-containing carrier compound yielded in step b) is recycled to step a) to regenerate the alkylated sulfur- or selenium-containing carrier compound.
  • kits comprising the employed components and the uses thereof to produce an alkylated substrate utilising the regeneration of spent carrier compound.
  • the invention provides a method for the production of the alkyl carrier compound in its alkylated form (briefly referred to as“carrier production method” herein).
  • an alkylated product of interest i.e. the alkylated substrate
  • the use of the alkyltransferase in step b) to transfer the optionally substituted alkyl group from an alkylated carrier compound to the substrate allows the alkylation of the substrate to proceed at a high yield and/or a high specificity for the desired product.
  • the use of the alkylransferase in step a) allows the alkylated carrier to be regenerated, such that the alkylated carrier can be used in amounts which are significantly below stoichiometric amounts, i.e.
  • alkylation method of the present invention makes alkyl donors available as sources of an afkyl group which are commercially available at lower costs than alkyl carriers disclosed in the literature for enzymatically controlled alkylation reactions, such as SAM.
  • the alkylation method provided by the invention removes the limitations of the prior art by a simple enzymatic (re-)generating/recycling of the alkyl carrier compound functioning as a co-substrate of an alkyltransferase (e.g (re-)generating S-adenosyl methionine (SAM) from the spent S-adenosyl homocysteine (SAH)), which can be accomplished by a single regenerating enzyme.
  • the co-substrate can be used as a catalytic, i.e. sub-stoichiometric component for the enzyme- catalyzed alkylation.
  • the co-substrate is provided by a carrier compound for the alkyl group, whereas a separate alkyl donor is used as a source of the alkyl group.
  • Alkyltransferases such as methyltransferases can catalyse the dealkylation of a co-substrate/carrier compound such as S-adenosyl methionine (SAM) to form specifically alkylated products and a dealky!ated co-substrate/carrier compound such as S-adenosylhomocysteine (SAH) as side product ( Figure 1 ).
  • SAM S-adenosyl methionine
  • SAH S-adenosylhomocysteine
  • an S-alkyttransferase can realkylate a co- substrate/carrier compound using a cost effective alkyl donor (e.g. remethy!ation of S- adenosylhomocysteine (SAH) to S-adenosyl methionine (SAM) by using methyl iodide or S ⁇ methyl methionine).
  • a cost effective alkyl donor e.g. remethy!ation of S- adenosylhomocysteine (SAH) to S-adenosyl methionine (SAM) by using methyl iodide or S ⁇ methyl methionine.
  • SAH S- adenosylhomocysteine
  • SAM S-adenosyl methionine
  • a halide methyltransferase or S-methionine methyltransferase and further N-, C-, 0-, S-, or P- alkyltransferase enables the transfer of alkyl groups from an alkyl donor (e.g. methyl iodide or methyl chloride; or S-methyl methionine) to a substrate, using the co-substrate (e.g. S- adenosyl methionine (SAM)) merely as an alkyl transfer catalyst.
  • an alkyl donor e.g. methyl iodide or methyl chloride; or S-methyl methionine
  • co-substrate e.g. S- adenosyl methionine (SAM)
  • SAM co-substrate
  • Such enzyme cascades require only catalytic concentrations of the alkyl carrier compound and use a different source as stoichiometric alkyl donor.
  • the method of the present invention is broadly applicable and can, inter alia, be applied for b-methyiation of L- and D-a-amino acids, b-methylation of hydroxy acids, b-methylation of alcohols or b-methylation of amines, preferably for b-methylation of L- and D-a-amino acids.
  • b-methylation can also be referred to as 2- methylation.
  • the relevant reaction steps including the involved enzymes of b-methylation or 2-methylation are shown in Figure 46.
  • b-methylation or 2-methylation is relevant because stereoselective introduction of b-methyl groups is difficult using traditional methods of organic synthesis.
  • one advantage of the method of the present invention is a high stereo-selectivity. Furthermore, synthesis of /3-methyl amino acids enables further application of such compounds in medicinal chemistry and/or chemical biology studies.
  • the alkylation method defined above as a first aspect of the invention will be described in further detail. However, the following definitions also apply to the other aspects of the invention relating to the kit and use thereof, and, as far as the provision of the alkylated sulfur- or selenium-containing carrier compound is concerned, also to the carrier production method further discussed below.
  • An alkylation step (also referred to herein as alkylation reaction) is a transfer of an alkyl group from one molecule to another molecule.
  • the alkyl group which is transferred in the alkylation method or the carrier production method in accordance with the invention is an optionally substituted alkyl group, i.e. an alkyl group which may be non- substituted, or may comprise one or more substituents.
  • An alkyf group is a group which can be derived from an alkane by removal of a hydrogen atom from a carbon atom.
  • An alkylated compound such as the alkylated carrier compound or the alkylated substrate used in the methods of the present invention, is a compound to which an alkyl group has been transferred ⁇ i.e.
  • a dealkylated compound such as the dealkylated carrier compound used in the alkylation method of the present invention, is a compound from which an alkyl group has been transferred (i.e. a compound from which an alkyl group has been removed). Also in this regard, it will be appreciated by the skilled reader that alkyl groups may be present in the dealkylated compound which are not involved in the transfer of an alkyl group.
  • the alkylation steps which are carried out in the context of the present invention involve the use of an alky!transferase.
  • An alkyltransferase is an enzyme which catalyzes an alkylation reaction and thus accomplishes a transfer of an (optionally substituted) alkyl group from one compound to another.
  • the alkyltransferase used in step a) and the alkyltransferase used in the carrier production method transfers an optionally substituted alkyl group to a sulfur- or selenium containing carrier compound.
  • the alkyltransferase used in step b) transfers the optionally substituted alkyl group to a substrate.
  • the alkyltransferase used in step a) is also referred to as first alkyltransferase, primary alkyltransferase, regenerating enzyme/regenerating alkyltransferase or enzyme.
  • the alkyltransferase used in step b) is also referred to as second alkyltransferase, or secondary alkyltransferase, further alkyltransferase, or alkylating enzyme.
  • the alkylation step a) yields an alkylated sulfur- or selenium-containing carrier compound by the transfer of an optionally substituted alkyl group from an alkyl donor.
  • the alkyl donor as used in the context of the present invention acts as a source of an optionally substituted alkyl group.
  • the alkyl donor is consumed when its optionally substituted alkyl group is transferred to the sulfur- or selenium containing carrier compound, i.e. the alkyl donor is not regenerated.
  • a sufficient amount of the alkyl donor should be provided for carrying out the alkylation method in accordance with the invention, taking due account of the amount of substrate to be alkylated.
  • at least one equivalent of the alkyl donor is provided per equivalent of the optionally substituted alkyl group to be transferred to the substrate.
  • the optionally substituted alkyl group of the alkyl donor is transferred from the alkyl donor to a sulfur- or selenium-containing carrier compound to yield an alkylated sulfur- or selenium-containing carrier compound.
  • the sulfur- or selenium-containing carrier compound which has not yet been alkylated may be briefly referred to as “carrier compound” or as “carrier compound prior to alkylation” herein.
  • the alkylated sulfur- or selenenium containing carrier compound may be briefly referred to as“alkylated carrier compound” herein.
  • the alkylation step b) yields an alkylated substrate as defined herein and a dealkyiated sulfur- or selenium containing carrier compound.
  • the dealkyiated carrier compound can be considered as spent carrier compound.
  • the dealkyiated carrier compound resulting from step b) will generally correspond in its structure to the carrier compound prior to alkylation. In other words, in terms of the compound structure, there are typically no differences between the sulfur- or selenium-containing carrier compound prior to alkylation and the dealkyiated sulfur- or selenium-containing carrier compound.
  • the sulfur- or selenium-containing carrier compound which has been dealkyiated may therefore equally be referred to as “carrier compound”, or as“dealkyiated carrier compound” herein.
  • the alkylated carrier compound can be referred to as an intermediate product in the alkylation method in accordance with the invention which carries an optionally substituted alkyl group received from an alkyl donor to a substrate to be alkylated.
  • the alkylated carrier compound can also be referred to as a product which is capable of carrying an optionally substituted alkyl group received from an alkyl donor.
  • the alkylation method in accordance with the invention can be considered as involving a net reaction as the sum of steps a) and b) wherein the substrate is alkylated with the optionally substituted alkyl group provided by the alkyl donor.
  • At least a part of the dealkylated sulfur- or selenium containing carrier compound yielded in step b) is recycled to step a) in order to regenerate the alkylated sulfur- or selenium containing carrier compound.
  • the predominant amount i.e. more than 50 mo!%, based on the total amount of dealkylated carrier compound as 100 mol%) is recycled to step a) to regenerate the alkylated carrier compound
  • the dealkylated sulfur- or selenium containing carrier compound yielded in step b) may be completely recycled to step a). It will be understood that the regenerated alkylated carrier compound resulting from this recycling is available again as a reactant in step b) in the alkylation method in accordance with the invention.
  • Each sulfur- or selenium containing carrier compound ⁇ i.e. each molecule of the carrier compound) used in the alkylation method in accordance with the invention can therefore be subjected to the alkylation step a) multiple times. It will be appreciated that once the alkylation step a) has been accomplished, the resulting alkylated sulfur- or selenium- containing carrier compound undergoes step b) to allow its optionally substituted alkyl group to be transferred to the substrate and to complete a cycle by yielding the dealkylated carrier compound which can then again be subjected to the alkylation step a).
  • each sulfur- or selenium containing carrier compound molecule used in the alkylation method in accordance with the invention is subjected to the alkylation step a) on the average at least 2 times, preferably at least 5 times, more preferably at least 10 times, even more preferably at least 50 times, still more preferably at least 100 times, and most preferably at least 500 times. Due to the subsequent transfer of the optionally substituted alkyl from the alkylated carrier compound to the substrate in step b), this average number of alkylation steps can be conveniently calculated from the yield of the alkylated substrate provided by the alkylation method and the amount of carrier compound used.
  • the alkyltransferase of step b) may transfer at least 5, more preferably at least 10, even more preferably at least 50, still more preferably at least 100, and most preferably at least 500 alkyl groups to substrate molecules to be alkylated per sulfur- or selenium-containing carrier compound molecule used.
  • the carrier compound can be used in amounts in the alkylation method in accordance with the invention such that less than one molecule of the carrier compound is provided for each optionally substituted alkyl group that is to be transferred to the substrate.
  • step a) As a first alkylation step, followed by step b) as a second alkylation step, or to carry out step b) as a first alkylation step, followed by step a) as a second alkylation step.
  • step a) the carrier compound prior to alkylation may be used as a starting compound together with the alkyl donor and the S-alkyltransferase of step a).
  • the alkylation of the carrier compound to provide the alkylated carrier would proceed as the first step in the reaction system containing these components, and the alkylated carrier compound can subsequently be used in step b), advantageously in the same reaction system.
  • an alkylated carrier compound can be obtained or can be preliminarily synthesized in a separate procedure, and can then be used as a starting compound together with the N-. C-, 0-, S-, or P-alkyltransferase of step b) and the substrate to be alkylated.
  • the alkylation of the substrate and the dealkylation of the alkylated carrier would proceed as the first step in the reaction system containing these components, and the dealkylated carrier can subsequently be recycled to a step a) to regenerate the alkylated carrier compound.
  • the alkylation of step a) is the first alkylation step, followed by step b) as the second alkylation step.
  • the alkylation method in accordance with the invention may also be referred to as a method for alkylating a substrate which comprises the following steps::
  • step c) a regeneration step, wherein the S-specific alkyltransferase transfers an optionally substituted alkyl group from an alkyl donor to the dealkylated sulfur- or selenium-containing carrier obtained in step b) to regenerate an alkylated sulfur- or selenium-containing carrier compound,
  • each sulfur- or selenium-containing carrier compound molecule used in the method is subjected to steps b) and c) on the average at least two times.
  • the alkyl donor in of the regeneration step c) preferably has the same structure as the alkyl donor of step a).
  • the alkyl donor in recycling step c) does not relate to the same alkyl donor molecule.
  • the alkyl donor in recycling step c) relates to a newly added alkyl donor molecule.
  • Alkylation step a) is preferably the first alkylation step that is carried out prior to steps b) and c).
  • each sulfur- or selenium-containing carrier compound molecule used in the preferred method is subjected to steps b) and c) on the average at least 5 times, more preferably at least 10 times, even more preferably at least 50 times, still more preferably at least 100 times and most preferably at least 500 times.
  • the alkyltransferase of step b) may transfer at least 5, more preferably at least 10, even more preferably at least 50, still more preferably at least 100, and most preferably at least 500 alkyf groups to substrate molecules to be alkylated per sulfur- or selenium-containing carrier compound molecule used.
  • the alkylation method and the carrier production method in accordance with the invention can be carried out in vitro or in vivo.
  • the methods can be conveniently carried out at or around room temperature, e.g. in a temperature range of 10 to 40 °C, more preferably 15 to 30 X. If the method is carried out in vitro, it is preferably carried out in a cell free-system or alternatively in a buffer.
  • a celf-free system may be an extract from a cell, which expresses the aikyttransferases used in steps a) and b).
  • the cell extract can be obtained by lysis of the cell (cell-free lysate). If the method is carried out in a buffer, the alkyltransferases used in steps a) and b) can be obtained from a cell, for example by purification, and added to the buffer.
  • a typical buffer is a sodium phosphate buffer, such as a 100 mM sodium phosphate buffer with a pH of 8,0.
  • the alkylation method in accordance with the invention is carried out (as an in vitro method) by combining the alkyl donor, the sulfur- or selenium-containing carrier compound, the S-alkyltransferase used in step a), the N-, C-, 0-, S-, or P- alkyltransferase used in step b) and the substrate to be alkylated in a single reaction vessel, before allowing the reaction to proceed.
  • the method is carried out in the absence of S-adenosyl adenosine nucleosidase (EC 3.2.2.9), when S-adenosyl homocysteine is used as a carrier compound. If the method is carried out in vivo, the host cell is preferably S-adenosyl adenosine nucleosidase (EC 3.2.2.9) deficient.
  • the method may be carried out in the absence of the methionine adenosyl transferase (MAT) to avoid a competition with unlabelled S-adenosyl methionine (SAM), which is generated by native S-adenosyl methionine (SAM) production in the host cell.
  • the host cell used may be methionine adenosyl transferase (MAT) deficient. It may be further required express the S-adenosyl transporter in the host cell, which is methionine adenosyl transferase (MAT) deficient, to ensure viability of the host cell.
  • R 1 is selected from the group consisting of a carboxy group, hydrogen, a triazole and an ester group;
  • R 2 is selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy group;
  • R 3 and R 4 are independently selected from hydrogen and a hydroxy group, and are preferably a preferably a hydroxy group;
  • B 1 is an optionally substituted purine or thienoadenine.
  • the sulfur-containing carrier compound represented by formula (la) is preferably in L- configuration, such that the sulfur-containing carrier compound prior to alkylation (and after dealkylation) may be represented by the following formula (laa):
  • R 1 , R 2 , R 3 , R 4 and B 1 are as defined above.
  • the ester group represents a carboxylate ester group, wherein the hydroxy group of the carboxylic acid is replaced by an alkoxy group
  • the alkoxy group preferably contains 1 to 9 carbon atoms.
  • Examples of the alkoxy group include methoxy, ethoxy, propoxy, butoxy, phenoxy and benzyloxy.
  • a primary amino group represents an amino group, wherein one of the three substituents bound to the nitrogen atom is an organic group other than hydrogen
  • a secondary amino group represents an amino group wherein two of the three substituents bound to the nitrogen atom are organic groups other than hydrogen
  • a tertiary amino group represents an amino wherein the three substituents bound to the nitrogen atom are organic groups other than hydrogen.
  • the organic group is preferably an aliphatic hydrocarbon group, more preferably an alkyl group, having 1 to 5 carbon atoms.
  • Suitable examples of the secondary amino group include methylamino, ethylamino, propylamino, allylamino and butylamino, preferably methylamino.
  • Suitable examples of the tertiary amino group include dimethylamino, diethylamino, dipropylamino, and dibutylamino, preferably dimethylamino.
  • the optionally substituted purine may be substituted by one or more, such as one, two or three, substituents.
  • substituents for the optionally substituted purine are selected from the group consisting of an alky! group having 1 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, an oxo group, a halogen atom, a hydroxy group, an amino group, a secondary amino group, and a tertiary amino group.
  • R 1 is preferably selected from the group consisting of a carboxy group, hydrogen and a ester group
  • R 2 is preferably selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group
  • R 3 and R 4 are preferably a hydroxy group
  • B 1 is preferably an optionally substituted adenine.
  • the optionally substituted adenine may be substituted by one or more, such as one, two or three, substituents.
  • substituents for the optionally substituted adenine are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, and a halogen atom.
  • S-adenosyl homocysteine preferably N6-methyl S-adenosyl homocysteine, N6-ethyl S-adenosyl homocysteine, N6-propyl S-adenosyl homocysteine, and N6-isopropyl S-adenosyl homocysteine.
  • the alkylated sulfur-containing carrier compound is preferably a methylated sulfur-containing carrier compound.
  • the methylated sulfur-containing carrier compound include S- adenosy! methionine (SAM), 2-fluoroadenosyl methionine, and S-methyl-S-(5'-adenosyl)-3- thiopropylamine, N6-substituted S-adenosyl methionine, S-adenosyl N-acetylmethionine, S- adenosyl N,N,-dimethylmethionine, S-adenosyl methionine methylester, S-adenosyl methionine ethylester and S-adenosyl methionine phenylester.
  • SAM S- adenosy! methionine
  • 2-fluoroadenosyl methionine 2-fluoroadenosyl methionine
  • the sulfur-containing carrier compound prior to alkylation (and after dealkylation) is most preferably S-adenosyl homocysteine (SAH).
  • SAH S-adenosyl homocysteine
  • SAM S-adenosyl methionine
  • R 5 is selected from the group consisting of a methyl group which may be substituted, an ethyl group which may be substituted and a propyl group which may be substituted.
  • substituents of R 5 are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, an oxo group, a halogen atom, a hydroxy group, an amino group, a secondary amino group, and a tertiary amino group.
  • the substituents of R 5 are preferably a halogen atom, more preferably a fluorine atom.
  • the optionally substituted alkyl group of the alkylated selenium-containing carrier compound is covalently bound to the selenium atom of the selenoether moiety.
  • the alkylated selenium-containing carrier compound may represent an ion analogous to a sulfonium ion.
  • the selenium-containing carrier compound prior to alkylation (and after dealkylation) is preferably represented by the following formula (lc):
  • R 6 is selected from the group consisting of a carboxy group, a hydrogen, a triazole, and an ester;
  • R 7 is selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy;
  • R 8 and R 9 are independently selected from hydrogen and a hydroxy group, preferably a hydroxy group; and B 2 is an optionally substituted purine or thienoadenine.
  • the selenium-containing carrier compound represented by formula (lc) is preferably in L- configuration such that the selenium-containing carrier compound prior to alkylation may be represented by the following formula (lea):
  • R 6 , R 7 , R 8 , R 9 and B 2 are as defined above.
  • ester group, the primary amino group, the secondary amino group, the tertiary amino group, and the optionally substituted purine are as defined above for the sulfur-containing carrier compound of formulas (la) and (laa).
  • R 6 is preferably selected from the group consisting of a carboxy group, hydrogen and a ester group
  • R 7 is preferably selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group
  • R 8 and R 9 are preferably a hydroxy group
  • B 2 is preferably an optionally substituted adenine.
  • the optionally substituted adenine may be substituted by one or more, such as one, two or three, substituents.
  • substituents for the optionally substituted adenine are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, and a halogen atom.
  • the selenium-containing carrier compound prior to alkylation is more preferably selected from the group consisting of Se-adenosyl selenohomocysteine, thienoadenosyl selenohomocysteine, Se-(2-fluoroadenosyI) selenohomocysteine, Se-(5'-adenosyl)-3- selenopropylamine, N6-substituted Se-adenosyl selenohomocysteine, Se-adenosyl N-acetyl selenohomocysteine, Se-adenosyl N,N, -dimethyl selenohomocysteine, Se-adenosyl selenohomocysteine methylester, Se-adenosyl selenohomocysteine ethylester and Se- adenosyl selenohomo
  • the N6-substituted Se-adenosyl selenohomocysteine is an Se-adenosyl selenohomocysteine which is substituted at the N6-position with an aliphatic or aromatic hydrocarbon group having 1 to 12 carbon atoms.
  • N6-substitufed Se-adenosyl homocysteine examples include N6-methyl Se-adenosyl homocysteine, N6-ethyl Se-adenosyl homocysteine, N6-propyl Se-adenosyl homocysteine, N6-benzyl Se-adenosyl homocysteine, N6-isopropyl Se-adenosyl homocysteine and N6-2-naphty!methyl Se-adenosyl homocysteine, preferably N6-methyl Se-adenosyl homocysteine, N6-ethyl Se-adenosyl homocysteine, N6-propyl Se-adenosyl homocysteine, and N6-isopropyl Se-adenosyl homocysteine.
  • alkylated selenium-containing carrier compound examples include Se-adenosyl selenomethionine, thienoadenosyl selenomethionine, Se-(2-f!uoroadenosyl) selenomethionine, Se-methyl-Se-(5'-adenosyl)-3-selenopropylamine, N6-substituted Se- adenosyl selenomethionine, Se-adenosyl N-acetyl selenomethionine, Se-adenosyl N,N,- dimethyl selenomethionine, Se-adenosyl selenomethionine methylester, Se-adenosyl selenomethionine ethylester and Se-adenosyl selenomethionine phenylester.
  • the carrier compound prior to alkylation is S-adenosyl homocysteine (SAH).
  • SAH S-adenosyl homocysteine
  • SAM S-adenosyl methionine
  • the alkyltransferase used in step b) transfers the optionally substituted alkyl group by a SN2-type reaction or a radical reaction to the substrate.
  • Alky!transferases which transfer the optionally substituted alkyl group by a SN2-type reaction are preferred.
  • a SN2-type reaction is nucleophilic substitution, of which the skilled person is familiar with.
  • the a!kyltransferase which transfers the optionally substituted alkyl group to the substrate by a SN2-type reaction, is preferably selected from the group consisting of the carminomycin 4- O-methyltransferase DnrK ⁇ originates from Streptomyces peucetius,), the demethylrebeccamycin-D-glycose O-methyltransferase RebM (originates from Lechevalieria aerocolonigenes,), the S-adenosyi-L-methionine-dependent O-methyltransferase (SynOMT) (originates from Synechocystis sp.
  • the carminomycin 4- O-methyltransferase DnrK ⁇ originates from Streptomyces peucetius is preferably selected from the group consisting of the carminomycin 4- O-methyltransferase DnrK ⁇ originates from Streptomyces peucetius,
  • O-specific alkyltransferases are preferably selected from the group consisting of the carminomycin 4-O-methyltransferase DnrK (EC 2.1.1.292; originates from Streptomyces peucetius,), the demethylrebeccamycin-D-glycose O-methyltra nsferase RebM (EC 2.1.1.164; originates from Lechevalieha aerocolonigenes, ), the S-adenosyl-L-methionine-dependent O- methyltransferase (SynOMT) (EC 2.1.1.338 and EC 2.1.1.339; originates from Synechocystis $p.
  • DnrK carminomycin 4-O-methyltransferase DnrK
  • DnrK demethylrebeccamycin-D-glycose O-methyltra nsferase RebM
  • SynOMT S-adenosyl-L-methionine-dependent
  • N-specific alkyltransferases are preferably selected form the group consisting of the phenazine-1 -carboxylate N-methyltransferase PhzM (EC 2 1.1 327; originates from Pseudomonas aeruginosa), the dTDP-3-amino-3,4,6-trideoxy-alpha-D-glycopyranose N,N- dimethyltransferase DesVI (EC 2.1.1 ,234; originates from Streptomyces fradiae), the NodS N-methyltransferase (EC 2.1.1.-; originates from Bradyrhizobium japonicum), originates from Bradyrhizobium japonicum, N-specific), the dTDP-3-amino-3,6-dideoxy-alpha-D- glucopyranose N,N-dimethyltransferase TylM1 (EC 2.1.1.235 and EC 2.1.1.236; originates from Streptomyces fr
  • the alkyltransferase used in step b) is selected from the group consisting of the histidine-specific methyltransferase EgtD (SEQ ID NO: 2), EC 2.1144), the inositol 4- methyltransferase (IMT, SEQ ID NO: 3) (EC 2.1.1.129), the putrescine N-methyl transferase (PMT, SEQ ID NO: 4) (EC 2.11.53), the methyltransferase SgvM (SEQ ID NO: 5, EC 2.1.1), the 8-demethylnovobiocic acid CS-methyttransferase NovO (SEQ ID NO: 6, EC 2.1.1.284) and the 5-thiohistidine N-methyltransferase OvoC (SEQ ID NO: 7, EC 2.1.1.-).
  • EgtD histidine-specific methyltransferase EgtD
  • IMT inositol 4- methyltransferase
  • the histidine-specific methyltransferase EgtD originates from Mycobacterium smegmatis, is N-specific and uses histidine as substrate.
  • the histidine methyltransferase EgtD is a SAM-dependent methyltransferase, which can catalyzes trimethylation of the alpha-amino group of histidine.
  • the resulting N-alpha-trimethyl histidine (TMH) is a precursor for ergothioneine biosynthesis in bacteria and fungi.
  • the inositol 4-methyltransferase originates from Mesembryanthemum crystallinum, is O- specific and uses inositol as substrate.
  • Inositol 4-methyltransferase (EC 2.11.129) converts inositol to ononitol.
  • O-Methyl inositols are plant osmoprotectants, which have antidiabetic, anti-cancer or anti-inflammatory effects in humans.
  • Inositol methyltransferases reflect the synthetic process of enzyme-mediated methylation in that they afford regiospecific alkylation of one out of six nearly equivalent hydroxyl groups. Chemical approaches for the same transformation include laborious multistep syntheses.
  • the putrescine N-methyl transferase (PMT) (EC 2.1.1.53) orginates from Anisodus tanguticus, is N-specific and uses diamine putrescine as substrate.
  • the utrescine N-methyl transferase (PMT, EC2.1.1.53) can catalyze the methylation of the diamine putrescine to form the first intermediate in the biosynthesis of alkaloids, including nicotine and tropanes. Transformation of primary amines to secondary amines by chemical synthesis is often hampered by the higher nucleophilicity of the secondary amine which results in overalkylation. Hence, secondary methyl amines are usually synthesized through indirect routes. In contrast, PMT avoids over-methyiation by way of its carefully tailored active site.
  • the methyltransferase SgvM can methylate the C3 position of alpha-ketonorvaline, among other substrates.
  • the 8-demethylnovobiocic acid C8-methyltransferase NovO (EC 2.1.1.284) originates from Streptomyces niveus, is C-specific and uses 8-demethylnovobiocic as substrate. NovO can methylate the sp2-hybridized carbon on the aromatic core of coumarins.
  • the alky!transferase used in step b) transfers the optionally substituted alkyl group by a radical reaction.
  • a radical reaction the alkyl group may be transferred in the form of a free radical, thus comprising an unpaired electron.
  • alkyltransferases are also referred to as radical alkyltransferases. These enzymes transfer methyl radicals to the substrates.
  • the radical aikyltransferase is preferably a methyltransferase, more preferably an S- adenosyl methionine (SAM) dependent methyltransferase.
  • SAM S- adenosyl methionine
  • the tryptophan 2-C-methyltransferase TsrM originates from Streptomyces laurentii, and is C- specific.
  • the P-methyltransferase originates from Streptomyces viridochromogenes, is P- specific and uses N-Acetyldemethylphosphinothricin tripeptide as substrate.
  • the valine methyltransferases PoyC originates from the bacterium symbiont of Theonella swinhoei pTSMAd , is C-specific and uses the peptide PoyA (SEQ ID NO: 8) as substrate.
  • Fosfomycin Biosynthesis Enzyme Fom3 originates from Streptomyces wedmorensis, is C-specific and uses (5'-cytidylyl)-2-hydroxyethylphosphonate as substrate.
  • GenK Gentamicin biosynthetic methyltransferase originates from Micromonospora echinospora, is C-specific and uses gentamicin X 2 as substrate.
  • SAM S-adenosyl methionine
  • SAH S-adenosyl homocysteine
  • 5dAdo 5-deoxy adenosine
  • SAM needs to be regenerated by two different systems: i) SAH could be recycled by as described above, for example by a halide methyl transferase and methyl halide; ii) Methionine could also be recycled by adenosyl-chloride synthase (chlorinase) using the (synthetic) co-substrate 5-chloro-5-deoxy adenosine (5-CI-dAdo), Overall this method uses three enzymes: a halide methyl transferase as aikyltransferase in step a), a radical methlytransferase in step b) and a chlorinase. The reaction consumes methyl halide, 5-CI- dAdo and substrate and produces methylated product, chloride and 5-deoxy adenosine (5dAdo).
  • step b) wherein the aikyltransferase used in step b), which transfers the optionally substituted alkyl group by a radical reaction, is a SAM-dependent aikyltransferase, preferably a SAM-dependent methyltransferase, and the sulfur-containing carrier compound is S-adenosyl homocysteine (SAH), step b) additional yields methionine.
  • SAH S-adenosyl homocysteine
  • step b) at least a part of the methionine yielded in step b) is regenerated by an adenosyl-chloride synthase (chlorinase), which synthesizes S-adenosyl-L-methionine (SAM) from methionine and a co-substrate.
  • chlorinase adenosyl-chloride synthase
  • SAM S-adenosyl-L-methionine
  • Converting enzyme convert a substrate into a product, or a presubstrate into a substrate.
  • the converting enzyme used in step c) is an enzyme that converts the beta-methylated substrate yielded in step b) to a beta-methylated product.
  • the converting enzyme used in step c) is preferably a transaminase or an isomerase.
  • the converting enzyme used in step d) is an enzyme that converts a presubstrate to the substrate that is alkylated in step b).
  • the converting enzyme used in step d) is preferably selected from the group consisting of a transaminase, an alcohol dehydrogenase and an alpha-keto acid decarboxylase.
  • a transaminase is an enzyme that catalyzes the amino transfer, for example between a a- keto acid and an amino acid, or between a primary amine and an aldehyde (see Figures 37 and 46 C).
  • An amino acid contains an amine (NH 2 ) group.
  • the amino acid is converted to a keto acid, and the keto acid is converted an amino acid.
  • the transaminase can use amino acids or proteins as substrate.
  • a transaminase uses the cofactor pyridoxal-phosphate (PLP).
  • An isomerase is an enzyme that catalyzes changes within one molecule.
  • An isomerease can convert one isomer to another.
  • An isomer has the same molecular formula but a different physical structure.
  • An isomer can be a structural isomers or stereoisomer.
  • Structural isomers have a different ordering of bonds and/or different bond connectivity from one another.
  • Stereoisomers have the same ordering of individual bonds and the same connectivity but the three-dimensional arrangement of bonded atoms differ.
  • Preferred isomerases are selected from the group consisting of racemases and epimerases.
  • Cis-trans isomerases are examples of enzymes catalyzing the interconversion of stereoisomers.
  • Intramolecular lyases examples of enzymes that catalyze the interconversion of structural isomers.
  • the reaction catalyzed by an isomerase is also called isomerization. See also Figure 37.
  • An alcohol dehydrogenase (ADH; EC 1.11.1 ) is an enzyme that catalyzes the conversion between alcohols and aldehydes or between alcohols and ketones.
  • the reaction catalyzed by ADH is preferably a redox reaction.
  • Alcohol dehydrogenase may use nicotinamide adeine dinucleotide (NAD+) as cofactor, which is reduced to NADH during the reaction. See also Figure 46 A and B.
  • An alpha-keto acid decarboxylase is an enzyme that catalyzes the conversion between ketones and alpha-keto acids. See also Figure 46 B and C.
  • the S-a!kyltransferase transfers an optionally substituted alkyl group from an alkyl donor to the sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound.
  • Examples of the optionally substituted alkyl group include a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyl group, a difluoromethy! group and a trifluoromethyl group. Further examples of the optionally substituted alkyl group include a 2-fluoroethyl group, 2,2-difluoroethyl group and 2,2,2-trifIuoroethyl group.
  • the alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group. More preferably, the atkyl group is a methyl group.
  • alkyl donor examples include methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, propargyl bromide, propargyl chloride, S-methyl methionine, Se-methyl selenomethionine, methyl sulfate, S-methyltetrahydrothiophene, S-methyl thietane, S-methyl ethylene sulphide, S-methyl di(monofluoromethyl) sulphide, S-methyl dimethyl sulphide, S ⁇ methyl 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid and S-methyl 3- (methylthio)propionic acid.
  • the alkyl donor being an oxygen-based alkyl donor is preferably represented by the following formula (II):
  • alkyl donor of formula (II) such as dimethylcarbonate reacts with the sulfur- or selenium-containing carrier compound such as SAH to produce an alkylated sulfur- or selenium-containing carrier compound such as SAM, R 11 XH such as methanol and carbon dioxide.
  • methyl iodide examples include methyl iodide, methyl bromide, methyl chloride and S-methyl methionine, still more preferably methyl iodide, methyl bromide and methyl chloride, most preferably methyl iodide.
  • Further examples of the most preferred methyl donor is monofluoromethyi iodide and Se-methyl selenomethionine.
  • the alkyl donor used in the alkylation method and the carrier production method according to present invention differs from the carrier compound used in these methods.
  • the alkyl donor is not S-adenosyl methionine (SAM)
  • the S-aikyltransferase transfers the optionally substituted alkyl group from the alkyl donor to the sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound; and further a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound.
  • the substrate (also referred to as acceptor) is an organic compound with a suitable atom suitable for alkylation, such as a sulfur atom, nitrogen atom, a carbon atom, oxygen atom or phosphorus atom.
  • the substrate according to the present invention preferably comprises at least one nucleophilic atom selected from the group consisting of a nucleophilic nitrogen atom, a nucleophilic carbon atom, a nucleophilic oxygen atom, a nucleophilic sulfur atom and a nucleophilic phosphorus atom. More preferably, it comprises at least one nucleophilic atom selected from the group consisting of a nucleophilic nitrogen atom, a nucleophilic carbon atom, and a nucleophilic oxygen atom.
  • the substrate preferably comprises a C-H acidic carbon-hydrogen bond or a group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group and phosphine group, more preferably a primary amino group, a secondary amino groups, a hydroxy group, an amide group, and a carboxy group.
  • the substrate may preferably also comprise a C-H acidic carbon-hydrogen bond and/or a group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a phosphonate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group, phosphine group, a carbonyl group and combinations thereof, more preferably a primary amino group, a secondary amino groups, a hydroxy group, an amide group, a carboxy group, a carbonyl group and combinations thereof,
  • nucleophilic carbon atom comprising a C-H acidic carbon-hydrogen bond
  • a carbon atom in alpha position to a carbonyl group or to a nitro group, or an aromatic carbon atom in ortho position to a hydroxy group may be mentioned.
  • Examples of the substrate prior to alkylation and/or of the alkylated substrate include a small molecule with a molecular mass of less than 1 kDa (1000 g/mol), primary metabolites, secondary metabolites, peptides such as ribosomal peptides, proteins, polysaccharides and nucleic acids.
  • the small molecule may have a molecular mass of less than 0,9 kDa (900 g/mol), preferably less than 0.8 kDa (800 g/mol). Small molecules as defined herein may also be primary metabolites and/or secondary metabolites.
  • Metabolite refers to intermediate or end products of the metabolism of a cell.
  • Primary metabolites may be directly involved in cell growth, development and reproduction. Secondary metabolites may be indirectly involved in these processes.
  • the primary metabolite is preferably selected from the group consisting of amino acids (such as histidine), nucleosides, nucleotides, lipids, carbohydrates.
  • the secondary metabolite is preferably selected from the group consisting of non-ribosomal peptides, polyketides, terpenes, alkaloids, phenylpropanoids, purtn- and pyrimidine-based metabolites, and antibiotics.
  • substrates prior to alkylation include histidine, inositol, putrescine, a-ketonorvaline, and coumarine.
  • substrates after alkylation include L- hercynine, ononitol, A/,W-dimethyl-1 ,4-butanediamine, R-3-methyl-2-oxovalerate, dihydroxy- 1-methylnaphthalene and ovothiol.
  • the substrate which is alkylated in step b) of the alkylating method in accordance with the invention differs from the carrier compound.
  • the substrate which is alkylated in step b) is not S-adenosyl methionine.
  • the alkylated substrate also referred to as the product or alkylated acceptor, is the substrate which comprises at least one optionally substituted alkyl group.
  • the at least one optionally substituted alkyl group of the alkylated substrate is covalently bound to a nitrogen atom, a carbon atom, an oxygen atom, a sulfur atom and/or a phosphorus atom of the substrate.
  • the alkylated substrate may be isolated and optionally purified,
  • the alkylated substrate may be isolated from the cell-free system or buffer if it was produced in vitro.
  • the alkylated substrate may be isolated from a celt if it was produced in vivo.
  • the cells may be lysed by any technique known to the skilled person, and purified from the supernatant after removing cell debris by centrifugation.
  • the alkylated substrate is further converted to an alkylated product.
  • the method is preferably used for b-methylation of L- or D-a-amino acids, L- or D-a-hydroxy acids, amines or alcohols, preferably for b-methylation of L- or D-a-amino acids.
  • the alkylated substrate yielded in step b) is a b-methylated substrate (for example b- methyl a-keto acid), which is further converted to a b-methylated product (for example b- methyl a-amino acid, or b-methyl a-hydroxy acid, or 2-methyl amine, or 2-methyl alcohol) by at least one additional step;
  • the b- ethylated product is preferably selected from the group consisting of a b-methyl-L-a-amino acid, a b-methyl-D-a-amino acid, a b-methyl-L-a-hydroxy acid, a b-methyl-D-a-hydroxy acid, a 2-methyl-amine and a 2-methyl-alcohol.
  • conversion step c) is a transamination wherein the converting enzyme is a transaminase that converts the b-methyl-a-keto acid to a b-m ethyl-ami no acid as the b-methylated product.
  • the method may comprise a further conversion step d ) after step b) and before step c), wherein step d ) is an isomarization step wherein an isomerase converts one isomere of the b- methylated et-keto acid to a different isomere. See also Figure 37 and 47.
  • the method described herein may comprise at least one additional step:
  • the substrate that is alkylated may be obtained from a presubstrate.
  • the presubstrate is a precursor of the substrate.
  • the presubstrate may be converted to the substrate by at least one converting enzyme in at least one additional conversion step d), which is preferably carried out before step b).
  • the presubstrate include amino acids, alpha-hydroxy acids, alpha-keto acids, amines, alcohols or ketones, preferably amino acids, alpha-hydroxy acids or ketones.
  • Alpha-hydroxy acids can additionally be obtained from alpha-keto acids.
  • Ketones can additionally be obtained from amines or alcohols.
  • Examples of the converting enzyme used in step d) are transaminase, alcohol dehydrogenase, or alpha-keto acid decarboxylase.
  • an amino acid as presubstrate can be converted to an alpha-keto acid as substrate by a transaminase as converting enzyme in step d).
  • An alpha-hydroxy acid as presubstrate can, for example, be converted to an alpha-keto add as substrate by alcohol dehydrogenase as converting enzyme in step d).
  • the alpha-hydroxy add presubstrate may additionally be obtained from an alpha-keto acid by an alcohol dehydrogenase as additional converting enzyme.
  • the method for alkylating a substrate in accordance with a particularly preferred embodiment of the invention comprises the following steps:
  • step b) wherein at least a part of the dealkyiated sulfur-containing carrier compound of formula (la) yielded in step b) is recycled to step a) to regenerate the alkylated sulfur-containing carrier compound of formula (la);
  • methyl halide is preferably methyl iodide or methyl chloride, more preferably methyl iodide.
  • the present invention further relates to a kit for alkylating a substrate comprising a S- alkyltransferase as defined herein above, a N-, C-, O-, S-, or P-alky!transferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above.
  • the S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention.
  • all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase comprised in the kit.
  • the N-, C-, 0-, S-, or P-alkyltransferase is an alkyltransferase as used in step b) of the alkylation method in accordance with the invention.
  • all definitions for the alkyltransferase used in step b) also relate to the N-, C-, 0-, S-, or P-alkyltransferase comprised in the kit.
  • the kit for alkylating a substrate may further comprise a substrate as defined herein above.
  • the substrate is a substrate prior to alkylation.
  • the kit for alkylating the substrate may further comprise a presubstrate and at least one converting enzyme, which converts the presubstrate to the substrate.
  • the presubstrate and the converting enzyme are as defined herein above.
  • the kit for alkylating a substrate is preferably substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3,2.2.9).
  • SAH S-adenosyl homocysteine
  • a kit substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) in the context of the present invention means that the kit comprises less than 5%, preferably less than 2%, more preferably less than 1 %, even more preferably less than 0.1% S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2 9).
  • the activity of the S-adenosyl homocysteine (SAH) nucleosidase may be less than 0.05% mol units per mg alkyltransferase, preferably 0.025% mol units per mg alkyltransferase, more preferably less than 0.013% mol units per mg alkyltransferase; and/or the activity of the S-adenosyl homocysteine (SAH) nucleosidase may less than 1 pmol/min per mg alkyltransferase, preferably 0.5 pmo!/min per mg alkyltransferase, more preferably less than 0.25 pmol/min per mg alkyltransferase.
  • the kit for methylating a substrate may comprise a halide methyl transferase as S-methyltransferase, the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase IMT, the putrescine N-methyltransferase PMT, or the 8-demethylnovobiocic acid C8- methyltransferase NovO as N-, C-, O-, S-, or P-methyltransferase; methyl iodide as a methyl donor and S-adenosyl homocysteine (SAH) as sulfur- or selenium-containing carrier.
  • a halide methyl transferase as S-methyltransferase
  • EgtD the histidine-specific methyltransferase EgtD
  • the inositol 4-methyltransferase IMT the putrescine N-methyltransferase PMT
  • the present invention also relates to the use of a S-alkyltransferase as defined herein above, a N-, C-, 0-, S-, or P-alkyltransferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above for the production of an alkylated substrate.
  • the S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention.
  • all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase used for the production of an alkylated substrate.
  • any substrate may be alkylated.
  • the substrates prior to alkylation as described herein above are preferred.
  • the alkylated substrate is preferably as described herein above.
  • the alkylated substrate is isotopically labeled.
  • an isotopically labeled alkyl donor as defined herein above is preferred.
  • the alkylated substrate is preferably produced in the absence of S-adenosyl homocysteine
  • the alkylated substrate may be isolated and optionally purified as described herein.
  • the present invention also relates to a method for producing an alkylated sulfur- or selenium- containing carrier compound, comprising an alkylation step, wherein a S-alkyltransferase as defined herein above transfers an optionally substituted alkyl group as defined herein above from an alkyl donor as defined herein above to a sulfur- or selenium-containing carrier compound as defined hereinabove, yielding an alkylated sulfur- or selenium-containing carrier compound.
  • the S-alkyftransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention.
  • alkyltransferase used in step a) also relate to the S-a!kyltransferase used in the method for producing an alkylated carrier compound.
  • the obtained alkylated sulfur- or selenium- containing carrier compound is preferably as defined herein above.
  • a methylated sulfur- or selenium-containing carrier compound is produced.
  • the optionally substituted alkyl group is a methyl group.
  • the alkylated sulfur- or selenium-containing carrier compound produced by the method is a methylated sulfur-containing carrier compound, more preferably S-adenosyl-methionine (SAM).
  • SAM S-adenosyl-methionine
  • the sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
  • the S-alkyltransferase which is preferably used in the method for producing S-adenosyl- methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum.
  • the alkyl donor is preferably methyl iodide, methyl bromide or methyl chloride, preferably methyl iodide or methyl chloride, more preferably methyl iodide.
  • a methionine S-methyltransferase preferably a methionine S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp. JA431, as S ⁇ alkyltransferase in the method for producing S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound.
  • SAM S-adenosyl-methionine
  • the alkyl donor is preferably S-methyl methionine or Se-methyl selenomethionine.
  • the method is for producing S-adenosyl-methionine (SAM) using S-adenosy! homocysteine (SAH), a halide methyl transferase as S-alkyltransferase and methyl iodide as alkyl donor.
  • SAH S-adenosyl-methionine
  • SAH S-adenosyl homocysteine
  • methionine S-methyltransferase as S-alkyltransferase and S-methyl methionine as alkyl donor.
  • the method for producing an alkylated sulfur- or selenium-containing carrier compound is preferably carried out in the absence of S-adenosyt homocysteine (SAH) nucleosidase (EC 3 2.2.9).
  • SAH S-adenosyt homocysteine
  • the absence of the S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2, 2.9) is preferred for the reasons explained herein above.
  • the alkylated carrier compound may be produced in vitro or in vivo.
  • a cell-free system may be an extract from a cell, which expresses the S-alky!transferase.
  • the cell extract can be obtained by lysis of the cell.
  • the S-a!kyltransferase is obtained from a cell, for example by purification, and added to the buffer.
  • a typical buffer is a sodium phosphate buffer, such as a 100 mM sodium phosphate buffer with a pH of 8.0.
  • the alkyl donor, the sulfur- or selenium-containing carrier compound and the S- alkyltransferase are typically combined in a single reaction vessel, before allowing the reaction to proceed.
  • the method is carried out in vivo, it is preferably carried out in a host cell.
  • the host cell is preferably a non-human or non-animal cell, more preferably a bacterial cell, plant cell or fungal cell. Particularly preferred bacterial cells are E.coli and C. Giutamicum.
  • the skilled person is familiar with suitable host cell and expression systems.
  • a gene encoding an alkyltransferase of the present invention may be cloned into a vector under control of a promoter such as an inducible promoter. After induction of the promoter, the gene is expressed and the method can be carried out.
  • the method is carried out in the absence of S-adenosyl adenosine nucleosidase (EC 3.2.2.9), when S-adenosyl homocysteine is used as a carrier compound. If the method is carried out in vivo, the host cell is preferably S-adenosyl adenosine nucleosidase (EC 3.2.2.9) deficient.
  • the alkylated carrier compound may be isolated and optionally purified.
  • the alkylated carrier compound may be isolated from the cell-free system or buffer if it was produced in vitro. Alternatively, the alkylated carrier compound may be isolated from a ceil if it was produced in vivo.
  • the cells may be lysed by any technique known to the skilled person, and the alkylated carrier compound is purified from the supernatant after removing ceil debris by centrifugation.
  • the alkylated sulfur- or selenium-containing carrier compound may be isolated and optionally purified.
  • the obtained alkylated sulfur- or selenium-containing carrier compound is preferably as defined herein above.
  • a methylated sulfur- or selenium-containing carrier compound is produced.
  • the optionally substituted alkyl group is preferably a methyl group.
  • the alkylated sulfur- or selenium-containing carrier compound produced with the kit is a methylated sulfur-containing carrier compound, more preferably S-adenosyl-methionine (SAM).
  • SAM S-adenosyl-methionine
  • the sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
  • the S-alkyltransferase which is preferably comprised in the kit for producing S-adenosyl- methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound, is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum.
  • the alkyl donor comprised in the kit may be methyl iodide, methyl bromide or methyl chloride, preferably methyl iodide or methyl chloride, more preferably methyl iodide.
  • a methionine S-methyltransferase preferably a methionine
  • S-methyltransferase from a rhodobacter more preferably from Rhodobacter sp. JA431, as S- alkyltransferase is comprised in the kit for producing S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound.
  • SAM S-adenosyl-methionine
  • MSMT methionine S- methyltransferase
  • the alkyl donor comprised is preferably S- methyl methionine or Se-methyl selenomethionine.
  • the kit for producing an alkylated sulfur- or selenium-containing carrier compound is preferably substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC).
  • SAH S-adenosyl homocysteine
  • SAH S-adenosyl homocysteine
  • the present invention also relates to the use of a S-alkyltransferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above for the production of an alkylated carrier compound.
  • the S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention.
  • all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase used for the production of an alkylated substrate.
  • the present invention further relates to the use of a kit as defined herein above for the production of an alkylated carrier compound.
  • the optionally substituted alkyl group is preferably a methyl group. It is preferred that the alkylated sulfur- or selenium-containing carrier compound produced is a methylated sulfur- containing carrier compound, more preferably S-adenosyl-methionine (SAM).
  • SAM S-adenosyl-methionine
  • the sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
  • the S-alkyitransferase which is preferably used for the production of S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound, is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum. If a halide methyl transferase is used, the alkyl donor is preferably selected from methyl iodide, methyl bromide and methyl chloride, more preferably methyl iodide or methyl chloride, still more preferably methyl iodide.
  • a methionine S-methyltransferase preferably a methionine
  • the alkylated substrate is preferably produced in the absence of S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9).
  • SAH S-adenosyl homocysteine
  • the alkylated carrier compound may be isolated and optionally purified.
  • the alkylated carrier compound may be isolated from the cell-free system or buffer if it was produced in vitro. Alternatively, the alkylated carrier compound may be isolated from a cell if it was produced in vivo.
  • the cells may be lysed by any technique known to the skilled person, and the alkylated carrier compound is purified from the supernatant after removing cell debris by centrifugation.
  • an alkyl carrier compound can be efficiently (re-)generated using the inventive system.
  • Example 1 demonstrates that several methyltransferases with N-, C- or O-specificity (EgtD, IMT; PMT, SgvM and NovO), which catalyze methylation of a variety of specific substrates using S-adenosyl methionine (SAM) as co-substrate, can be combined with a S-specific halide methyitransferase and methyl iodide as stoichiometric alkyl donor to regenerate the catalytic methyl carrier S-adenosyl methionine (SAM).
  • SAM catalytic methyl carrier S-adenosyl methionine
  • Example 1 demonstrates that the methyitransferase for alkylating a substrate and its substrates are not limiting for the system. Thus, it can be assumed that the system is applicable to any alkyltransferase-substrate combination.
  • Example 1 further demonstrates that S-adenosyl methionine (SAM) can be generated, i.e. produced, from S-adenosyl homocysteine (SAH) using the S-specific halide methyitransferase and methyl iodide as alkyl donor.
  • SAM S-adenosyl methionine
  • Removal of S-adenosyl homocysteine nucleosidase can be achieved either by re-purifying the employed alkyltransferase or by deleting the gene encoding the S-adenosyl homocysteine nucleosidase in the host cell, which is used for expressing the employed alkyltransferases.
  • Example 1 further demonstrates that S-adenosyl methionine (SAM) can be generated, i.e. produced, from S-adenosyl homocysteine (SAH) by using the S-specific methionine S- methyltransferase and S-methyl methionine as alkyl donor.
  • SAM S-adenosyl methionine
  • SAH S-adenosyl homocysteine
  • an S-aikyltransferase and an alkyl donor can be used to generate S-adenosyl methionine (SAM) from S-adenosyl homocysteine (SAH). It can be expected that this system is also applicable to other alkyl carrier compounds, for example selenium-based SAH/SAM derivatives or methionine-based alkyl carrier compounds. It has been further plausibly demonstrated that the S- alkyltransferase, alkyl donor can be employed in the alkylation of a substrate by a further alkyltransferase to regenerate the co-substrate of the second alkyltransferase, i.e. the alkyl carrier compound.
  • System 3 In vivo isotope labelling using methionine or S-methyl methionine as methyl donor.
  • Labelled S-methyl methionine (SMM) is synthesized and fed to cells (e.g. E coir).
  • One methyl group of this metabolite is transferred to homocysteine by the enzyme S- methylmethionine-homocysteine S-methyltransferase (EC 2.1.1.10).
  • Both methionine equivalents are activated to SAM by methionine adenosyl transferases (MAT, EC 2.5.16) and used for substrate methylation by an overproduced MT.
  • MAT methionine adenosyl transferases
  • Radical-MTs catalyse very difficult chemical reactions. Exploitation of such enzymes for biocatalytic application will significantly broaden the scope of enzyme-based synthesis.
  • This cascade provides a methodology to use radicai-MTs without the need for SAM as a stoichiometric reagent. Instead, the reaction is driven by the relatively simple reagents methyl halide and 5-Cl-dAdo.
  • step b) an alkylation step wherein a N-, G-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound, and wherein at least a part of the dealkylated sulfur- or selenium-containing carrier compound yielded in step b) is recycled to step a) to regenerate the alkylated sulfur- or selenium-containing carrier compound
  • step a) a S-alkyltransferase transfers the optionally substituted alkyl group from the alkyl donor to a sulfur- containing carrier compound, yielding an alkylated sulfur-containing carrier compound;
  • a N-, C-, 0-, S-, or P-alkyitransferase transfers the optionally substituted alkyl group from the alkylated sulfur-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur-containing carrier compound,
  • R 6 is selected from a group consisting of a carboxy group, a hydrogen, a triazole, and an ester;
  • R 8 and R 9 are independently hydrogen or a hydroxy group, preferably a hydroxy group
  • B 2 is an optionally substituted purine or thienoadenine.
  • step b) is an S-adenosyl methionine (SAM) dependent alkyltransferase.
  • SAM S-adenosyl methionine
  • alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT), thioether methyltransferases and N-methyl proline methyltransferase, N-dimethyl glycine methyltransferase and N-dimethyl beta-alanine N-methyltransferase, wherein the thioether methyltransferase is preferably selected from methionine 8- methyltransferase (MSMT)
  • HMT halide methyl transferases
  • MSMT methionine 8- methyltransferase
  • alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT) and methionine S-methyltransferase (MSMT).
  • HMT halide methyl transferases
  • MSMT methionine S-methyltransferase
  • alkyltransferase used in step a) is a ca rboxyl m eth yltransferase , preferably a indole-3-acetic acid carboxyl methyltransferase, Trigonelline synthase, Gibberellic acid methyltransferase, salicylic acid carboxyl methyltransferase, Jasmonic acid carboxyl methyltransferase, or Caffeine synthase.
  • HMT halide methyl transferase
  • step b) transfers the optionally substituted alkyl group by a SN2-type reaction
  • the alkyltransferase used in step b) is selected from the group consisting of the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase (IMT), the putrescine N-methyl transferase (PMT), the methyltransferase SgvM, the 8- demethylnovobiocic acid C8-methyltransferase NovO and the 5-thiohistidine N- methyltransferase OvoC.
  • the alkyltransferase used in step b) is selected from the group consisting of the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase (IMT), the putrescine N-methyl transferase (PMT), the methyltransferase SgvM, the 8- demethylnovobiocic acid C8-methyltransferase NovO and the 5-thiohistidine N- methyltrans
  • alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group, preferably a methyl group.
  • alkyl donor is selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, propargyl bromide, propargyl chloride, S-m ethyl methionine, Se-methyl selenomethionine, methyl sulfate, 8-methyl tetrahydrothiophene, S- methyl thietane, S- methyl ethylene sulphide, S-methyl di(monofluoromethyl) sulphide, S-methyl dimethyl sulphide, 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid, S-methyl 3- (methylthio)propyl donor, methyl methyl methyl methyl methyl
  • R 10 is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyt group, a butyl group, a monof!uoromethyl group, a difluoromethyl group and a trifluoromethyl group, preferably a methyl group;
  • R 10 is selected from an alkyl group or an aryl group, preferably a methyl group or ethyl group,
  • the substrate comprises a nucleophilic atom selected from the group consisting of a nitrogen atom, a carbon atom, an oxygen atom, a sulfur atom and a phosphorus atom
  • kit according to item 50 further comprising a substrate as defined in any of items 44 to 46.
  • a method for producing an alkylated sulfur- or selenium-containing carrier compound comprising an alkylation step, wherein a S-alkyltransferase as defined in any of items 1 , 16 and 19 to 25 transfers an optionally substituted alkyl group as defined in any of items 1 , 36 to 39 from an alkyl donor as defined in any of items 1 and 35 to 43 to a sulfur- or selenium-containing carrier compound as defined in any of items 1 and 3 to 14, yielding an alkylated sulfur- or selenium-containing carrier compound.
  • Figure 26 Measured and calculated HRMS of compound shown in Figure.
  • Figure 27 Measured and calculated HR-MS of f?-3-methyl-2-oxovalerate.
  • Figure 31 General scheme of a catalytic alkylation system with regeneration of the alkylated carrier.
  • FIG. 34 Extracted ion chromatogram (EIC) of isotope labeled ovothiol A produced by the cascade methyl transfer.
  • Figure 36 natural biosynthetic pathway of b-Me AA.
  • the existing examples includes jS-Me Glu, j3-Me Trp, b-Me Phe, b-Me Leu.
  • Figure 37 Strategy on stereo-selective /J-methylation of amino acids in this study.
  • Amino acid is converted to a-keto acid by a transaminase (TA)
  • TA transaminase
  • HMT halide methyltransferase
  • Methylated a-keto acid is then converted to b-methyl amino acid by TA.
  • Methyltransferases can transfer a methyl cation from SAM to C-. N-, 0-, P-, S-nucieophiles.
  • the sulfontum moiety of SAM is kineticatly stable so that alkyl transfers only occur in the confinement of enzyme active sites.
  • the thermodynamic reactivity of SAM is high enough to make methylation of almost any nucleophile (N, C, O, P, S) favorable (AG ⁇ 0).
  • HMT/EgtD cascade HMT/EgtD cascade.
  • HMT was combined with EgtD, a histidine methyltransferase from Mycobacterium smegmatis.
  • This SAM-dependent methyltransferase catalyzes trimethylation of the ct-amino group of histidine following a cooperative mechanism.
  • the resulting Na-tri methyl histidine (TMH) is a precursor for ergothioneine biosynthesis in bacteria and fungi.
  • TMH Na-tri methyl histidine
  • Time-dependent product formation was monitored by HPLC (B, Figure 1 ), In a reaction containing 1 mM histidine, 6 mM methyl iodide, 50 mM SAH, and 10 mM of EgtD and HMT thermo in a 100 mM phosphate buffer at pH 8, we observed TMH formation at an initial rate of 0.68 mM/min (C, Figure 2). After two hours this rate decreased significantly, and after 23 hours we determined that about 15 % of histidine was converted to TMH (Table 1 , entry 1 ). This conversion indicated that every SAH molecule was remethylated by HMT nine times. Importantly, a reaction tacking either of the two enzymes produced no detectable TMH ⁇ B, Figure 2). This experiment provides the first demonstration that SAH can serve as a catalytic methyl carrier in enzyme catalyzed reactions.
  • HMT and EgtD in an E coli strain that is SAH nucleosidase-deficient (KEIO collection, strain JWQ155-1 ).
  • KEIO collection strain JWQ155-1
  • T7 RNA polymerase gene by lysogenesis (Supplementary information).
  • the resulting strain produced HMT and EgtD with a similar efficiency as BL21(DE3) cells.
  • Inositol 4-methyltransferase from Mesembryanthemum crystallinum (IMT, EC 2.1.1.129) converts inositol to ononitol (Table 2, entry 3).
  • O-Methyl inositols are plant osmoprotectants, that also have antidiabetic, anti-cancer or anti-inflammatory effects in humans.
  • Inositol methyltransferases reflect the synthetic process of enzyme-mediated methylation in that they afford regiospecific alkylation of one out of six nearly equivalent hydroxyl groups. Chemical approaches for the same transformation include laborious multistep syntheses,
  • Putresdne N-methyl transferases catalyze the methylation of the diamine putresdne to form the first intermediate in the biosynthesis of alkaloids, including nicotine and tropanes (Table 3, entry 4). Transformation of primary amines to secondary amines by chemical synthesis is often hampered by the higher nucleophilicity of the secondary amine which results in overalkylation. Hence, secondary methyl amines are usually synthesized through indirect routes. In contrast, PMT avoids over-methylation by way of its carefully tailored active site.
  • C-Methylating enzymes are notable for their abilities of asymmetric alkylation of the beta- carbon of alpha-keto acids (Table 3, entry 5). This activity is of particular interest, since direct asymmetric alkylation alpha to ketones is stilt a challenging objective for synthetic methods.
  • 26 As a test enzyme we examined the methyltransferase SgvM from Streptomyces griseoviridis, which can methylate the C3 position of alpha- ketonorvaline, among other substrates.
  • Another class of C-methylating enzymes is represented by the enzyme NovO, which can methylate the sp2-hybridized carbon on the aromatic core of coumarins (Table 3, entry 6J.
  • Enzyme cascades consisting of HMT and O-, N- or C-methylating methyl transferases represent simple and robust methylation platforms. Fueled with the off-the-shelf reagent methyl iodide and catalytic concentrations of SAM or SAH these cascades can exploit the extraordinar regio- » chemo-, and stereoselectivity of naturally evolved SAM- dependent methyltransferases for in vitro methylation reactions. Based on the results, it is reasonable to expect that radicai-SAM methyltransferases can also be combined with the HMT-dependent SAM regeneration system. Similarly, it can be expected that this methylation technology could be extended to utilize SAM derivatives as a general approach for enzyme catalyzed alkyl transfers.
  • the gene encoding enzymes were was codon-optimized and synthesized by General Biosystems, Inc. (Morrisville, NC, USA) after which it was cloned (using Ncol/Xhol sites) into the pET-28a plasmid. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions. The gels were stained with Coomassie brilliant blue.
  • E. coli BL21(DE3) cell pET-28a plasmids containing genes were transformed using standard heat- shock protocols for chemically competent E, coli BL21(DE3) cells.
  • E. coli cells containing the plasmid were collected from LB-AGAR plates with Chloramphenicol (35 pg/ml ⁇ & Kanamycin (50 pg/ml) and used to inoculate LB medium with Chloramphenicol (35 pg/ml) & Kanamycin (50 pg/ml) (20 ml).
  • E. coli strains pET-28a plasmids containing genes were transformed by electroporation. Cells containing the plasmid were collected from LB-AGAR plates with Kanamycin (50 pg/ml) and used to inoculate LB medium. After inoculation, expression was done by following the same protocol as that of E. coli BL21(DE3) cell.
  • lysis buffer 50 mM phosphate, 300 mM NaCI, 10 mM imidazole, pH 8.0. Cells were disrupted by sonication for 3 x 60 s with Branson sonifier 450 (output control 5, 50% duty cycle). Lysates were centrifuged at 10,000 x g for 1.0 h at 4 °C. The cleared lysate was mixed with 1 ml of Ni" NTA agarose at 4 °C for 20 min and loaded onto a column. The agarose beads were washed with 10 ml sodium phosphate buffer containing 10 mM and 20 mM imidazole.
  • the protein was eluted in a lysis buffer solution containing 250 mM imidazole. Protein containing fractions were collected and dialyzed against dialysis buffer (100 mM phosphate, pH 8) aliquoted and stored at -80 °C.
  • Plasmid pKD46 was transformed to cell strain from the KEIO collection (CGSC #: 8422, MTA/SAH nucleosidase knockout) by electroporation to delete the kanamycin resistant cassette from chromosome. The cells was inoculated onto plate with ampiciliin and incubated at 30 °C. Single colony was picked and suspended in 50 pi LB-medium and then spreaded onto plate without any antibiotic and incubated at 42 °C overnight. The incubation at 42 °C was repeated for three times.
  • Lysogenization was done by following the protocol of the Novagen ADE3 Lysogenization Kit provided by the producer. Firstly, cells from single colonies were grown in LB medium supplement with 0.2% maltose, 10 mM MgS0 4 at 37 °C to an OD eoo of 0.5. Secondly, 10 pi cultures were mixed with 5 pi of mixture containing 10 ® pfu ADE3. 10 ® pfu Helper Phage, and 10 ® pfu Selection Phage and incubated at 37 °C for 20 min. Then the mixtures were spreaded onto LB plates and plates were incubated overnight at 37 °C. Colonies were randomly picked from plates and used to prepare cell cultures for further verification, storage and protein production. Cascade reaction assay
  • reaction mixture was dissolved in 600 pL D 2 Q for 1 H NMR recording.
  • reaction mixture was dissolved in 600 pL D 2 0 for 1 H NMR recording. Conversion was determined based on integral of protons from both substrate and product,
  • reaction containing TOO mM sodium phosphate buffer, 2 mM N-methyltra nsferase , 20 mM SAH, 8 mM methyl iodide, 10 mM HMT and 20 mM PMT were incubated at 25°C for 72 hours and lyophilized. Reaction mixture was dissolved in 600 pL D 2 Q for 1 H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
  • reaction mixture containing 100 mM sodium phosphate buffer, 5 mM substrate, 50 pM SAH, 10 mM methyl iodide 10 mM HMT and 40 mM IMT were incubated at 25 °C for 24 hours. Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Supernatant was centrifuged and lyophilized. Reaction mixture was dissolved in 600 pL D 2 0 for 1 H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
  • HMT_NovO cascade and 1 H NMR analysis 1 ml reaction containing 100 mM sodium phosphate buffer, 5 mM substrate, 50 mM SAH, 10 mM methyl iodide 10 mM HMT and 40 mM NovO were incubated at 25 °C for 24 hours. Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Supernatant was centrifuged and lyophilized. Reaction mixture was dissolved in 600 pL D 2 0 for 1 H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
  • Example 2 S-adenosyl-L-methionine regeneration by the enzyme S-methyl methionine methyl transferase (MSMT)
  • SAM S-adenosyl-L-methionine
  • MTs S-adenosyl-L-methionine dependent methyltransferases
  • SAM S-adenosyl-L-methionine
  • MTs catalyze the methyl group transfer from SAM to C-, 0-, N-, or S-centered nucleophiles and substrates of MTs include nucleic acids and proteins, as well as a lot of secondary metabolites.
  • Those transfers are normally highly chemo- and regio-selective.
  • C-methyltransferase the transfers are stereo-selective as well. Therefore, MTs has significant potential for application in biotechnology because they can be used for methylation of synthetic compounds and natural products, which are often important for the bioactivities of those compounds.
  • MSMT Methionine S-methy!transferase
  • Rhodobacter sp Rhodobacter sp, JA431, and validated the function in vitro.
  • Kinetic parameters have been measured under different conditions. Jc cat was measured to be around 10 min 1 . K u was observed to be 9.9 ⁇ 0.6 mM for methionine and 1.5 ⁇ 0.2 mM for SAM.
  • a cascade reaction ⁇ Figure 32 was set up to verify the concept. 250 pi of reaction were run under 50 mM of phosphate buffer at pH 8.0. Final concentrations of Chemicals and enzymes were as following: NaCI 20 mM, SAH 100 mM, S-methyl methionine 10 mM, TCEP 2 mM, 5-thioIhistidine 1 mM, MSMT and OvoC 10 mM. In negative control, no MSMT was used. In positive control, 300 uM of SAM were used instead of MSMT and SAH. Thiol of ovothiol A was modified with bromo-coumarin. The production of ovothiol A was monitored by detecting the BMC derivative by reverse phase HPLC.
  • OvoC transferred a methyl group from SAM and produced ovothiol A in positive control.
  • Around 10 mM of ovothiol A has been produced by the cascade reaction.
  • same signal can be observed in the negative control reaction without MSMT.
  • ovothiol A in the cascade reaction can be converted from purified enzymes bounded SAM.
  • LC-MS coupled with isotope labelled SMM was performed. The reaction conditions were as same as the reaction before. Two reactions containing either SMM or SMM-CD3 were run in parallel for UPLC-MS measurement.
  • Ovothiol A standard was injected as well to get the standard retention time of extracted ion chromatogram (EIC) ( Figure 2 of Appendix 2 ⁇ Experiment 2)).
  • MSMT was shown to accept Se-methyl selenomethionine (SeMSeM) as methyldonor to produce SAM from SAH ( Figure 47).
  • Example 3 Applicability of the HMT/MT technology to other aikyltransferase
  • the system such as the HMT/MT system, is also applicable to reactions that transfer chemical groups other than methyl groups, including but not limited to stable methyl isotopologues, ftuoromethyl, ethyl or propargyl groups.
  • Empirical analysis and computational modelling of the HMT/MT technology can be characterized by empirical quantification of time- dependent enzyme activities, the accumulation of protein modifications, and concentrations of substrates, products, intermediates, SAH and SAM. This data can be used to construct a numeric model describing ail kinetic aspects of HMT/MT cascade reactions.
  • KinTek Explorer Chemical Kinetics Software can be used. Based on this model predictions can be made as to how the cascade efficiencies change as a response to improving individual c ⁇ ants, or to changing substrates.
  • the above quantitative analysis will identify cascade components and reaction conditions that allow a further optimization.
  • the information can be used for the following:
  • immobilized enzymes also simplifies the downstream process, because the aqueous solution containing methylated product, volatile buffer salts, sodium iodide and catalytic amounts of SAH/SAM (1 %) can be filtered of and lyophifized to obtain highly pure product as the acetate salt.
  • Methyl iodide has proven an excellent methyl donor for enzyme catalysed methylation.
  • methyl chloride may be favourable.
  • Example 4 Direct stereo-selective b-methylation of amino acids by enzymatic cascades linking two co-factor regeneration systems
  • Methylation is a minor modification, but has profound effect on bioactivities of different molecules in medicinal chemistry. This effect has been given a name of“The Magic Methyl Effect” by medicinal chemist because introducing a methyl group to a small molecule can result in up to 2000-fold boost in potency. Due to this effect, methylation reaction has received remarkable interest in organic synthesis over the past years. Despite the effort scientists have made, asymmetric methylation of non-activated methylene C-H bond remains unsolved.
  • /8-methyl amino acids (0-Me AA) is a series of non-canonical amino acids, which have been found in non-ribosomal peptides (NRP) and other natural products (Figure 35).
  • NRP non-ribosomal peptides
  • Figure 35 The new stereo center of 0-carbon, together with a-carbon, constrains confirmation of the side chain and affect the properties of the molecules that 0-Me AAs compose. It is not surprising that many nature products containing 0-Me AAs are bioactive and some of them, such as daptomycin, are being used in clinic. Furthermore, this effect has been demonstrated in some examples of synthetic peptides, wherein introducing b-Me AAs can prolong the biological activity and result in higher potency and selectivity.
  • jS-Me AAs are produced via four different approaches.
  • the most common approach comprises three steps.
  • amino acid is converted to cr-keto acid by a transaminase (TA) with a-keto glutarate as amine acceptor.
  • TA transaminase
  • er-keto acid is asymmetrically methylated by a SAM-dependent methyltranferase (MT).
  • MT SAM-dependent methyltranferase
  • methylated keto acid is converted to jS-Me AAs by the same transaminase of the first step.
  • the existing products of this approach include b-Me arginine, b-Me phenylalanine, b-Me tryptophan, b-Me glutamate and b-Me leucine.
  • Enzymatic cascade would offer great economic benefit by avoiding the purification of unstable intermediate and reduce the usage of organic solvents during the purification process. See also Figure 36.
  • AA is converted to a-keto acid by a transaminase (TA).
  • TA transaminase
  • PLP- dependent transaminases has a ping-pong mechanism
  • ammonia of AA will be trapped within the active site as Pyridoxamine-S'-phosphate if there is no cr-keto glutarate as ammonia acceptor, Meanwhile, the released cr-keto acid can be irreversibly methylated by SAM dependent MT.
  • Pyridoxamine-5’-phosphate bound TA would take b-Me- s-keto acid as substrate and converted to b-Me AA
  • the cofactor PLP is regenerated.
  • HMT halide methyltransferase
  • the diastereoisomer was produced because of non- enzymatic racemization of b-Me indolpyruvate and both of the enantiomers are substrates of MarG. In principle, it is shown that our design is practical and PLP is tolerant of methyl iodide under this condition.
  • branched chain amino acids transaminase iLvE
  • SgvM methyltransferase
  • L-allylglycine ( ⁇ ) was methylated with 80% of conversion.
  • L-2-amino-5-methyl-Hex-4-enoic acid (8) was methylated with conversion of 70 %.
  • Compound 8a is a residue of Cyciomarins peptide.
  • L-methionine (9) was substrate as well and conversion was moderate of 55%. In all the cases, diastereomer ratio was not determined because WMR signals were overlapping.
  • HMT monofluoromethyl group
  • E coli Amtn E3 cell and purified both proteins by Ni' -affinity chromatography.
  • SAH sodium phosphate
  • SAM F-SAM
  • F-TMH monofluoromethyldimethyl histidine

Abstract

The present invention relates to a method for alkylating a substrate comprising an alkylation step a) wherein a S-alkyltransferase transfers an alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated carrier compound and an alkylation step b) wherein a N-, C-, O-, S- or P- alkyltransferase transfers the alkyl group from the alkylated carrier compound to the substrate, yielding an alkylated substrate and a dealkylated carrier compound, and wherein at least a part of the dealkylated carrier compound is recycled to regenerate the alkylated carrier compound. Furthermore, the invention relates to a method for producing the alkylated sulfur- or selenium-containing carrier compound. The invention also relates to kits for alkylating a substrate or for producing the alkylated carrier compound and the use of the components and kits for the production of an alkylated substrate or an alkylated carrier compound.

Description

Method for the biocatalytic alkylation of a substrate
INTRODUCTION
The present invention relates to a method for alkylating a substrate comprising an alkylation step a) wherein a S-alkyltransferase transfers an alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated carrier compound and an alkylation step b) wherein a N-, C-, O-, S- or P- alkyitransferase transfers the alkyl group from the alkylated carrier compound to the substrate, yielding an alkylated substrate and a dealkylated carrier compound, and wherein at least a part of the dealkylated carrier compound is recycled to regenerate the alkylated carrier compound. Furthermore, the invention relates to a method for producing the alkylated sulfur- or selenium-containing carrier compound. The invention also relates to kits for alkylating a substrate or for producing the alkylated carrier compound and the use of the components and kits for the production of an alkylated substrate or an alkylated carrier compound.
Alkylation, in particular methylation, is a common reaction in natural product biosynthesis and in signal transduction. Introduction of alkyl groups, in particular methyl groups, onto small and large biomolecules can change their physiochemical properties significantly. Therefore, alkylation, in particular methylation, is an important synthetic approach to optimize the potency and pharmacokinetic properties of therapeutic compounds. Although alkyl transfers are conceptionally simple, attachment of alkyl groups to specific functions on complex molecules is challenging. The development of synthetic methodologies to overcome this problem is an important frontier in organic chemistry. The large and rapidly growing number of known enzymes that can alkylate a broad variety of molecules with exquisite regio-, chemo- and stereospecificity, compounded with the maturing abilities to adapt the substrate scope of enzymes by design and selection, suggest that biocatalysis may play an important role in overcoming the synthetic challenges of late-stage alkylation (e.g. methylation). Despite this promise, the biocatalytic scope of alkyltransferases (e.g. methyltransferases) is limited because these enzymes require stoichiometric amounts of the expensive cosubstrates such as S-adenosyl methionine (SAM), i.e. at least one molecule of the cosubstrate has to be provided as a starting material for each alkyl group that is to be transferred to the desired product, In addition to being used for methylation, S-adenosyl methionine (SAM) is also used as a medicament
Preparative applications of enzyme-catalyzed alkylation is prohibited by the high cost of the stoichiometric co-substrate S-adenosyl methionine (SAM) (> 1000 Euro/1 g). Chemical synthesis has been considered as a possible source for S-adenosyl methionine (SAM). This approach is complicated by the fact that the last step in this synthesis - methylation of S- adenosyl homocysteine (SAH) - produces mixtures of the active (S,S)- and the inactive (F?,S)-isomers of S-adenosyl methionine (SAM), The introduction of chemoenzymatic approaches to produce diastereomerically pure S-adenosyl methionine (SAM) and SAM- derivatives has greatly broadened the scope of enzyme-catalyzed alkylation. However, these innovations did not address the fundamental problem of co-substrate stoichiometry. Thus, the requirement of SAM as a stoichiometric co-substrate is an unsolved problem in the industrial application of methyltransferase.
The first enzyme-catalyzed methylation reaction that dispels the stoichiometric requirement of SAM was published only recently (Mordhorst, S., Siegrist, J., Mueller, M , Richter, M., and Andexter, J. N. (2017) Angew Chem Int Ed Engl. 56, 4037 - 4041 ). This publication describes an in vitro reconstituted enzyme cascade that regenerates S-adenosyl methionine (SAM) from S-adenosyl homocysteine (SAH) mimicking part of the cellular adenosine metabolism. Briefly, after methyl transfer from SAM to product the cascade catalyzes the hydrolysis of SAH to adenosine (EC 3.3.1.1 ), phosphorylation of adenosine to ATP (EC 2.7.1.20, EC 2.7,4.6 or EC 2.7.1.40), and condensation of ATP with methionine to regenerate SAM (EC 2 5.1.6) (Figure 1 ). The chemical energy required for adenosine phosphorylation was provided by polyphosphates. This system was shown to turnover up to eleven times and marks an important step towards biocatalytic applications of MTs. On the other hand, a cascade consisting of six enzymes and 14 metabolites is inherently difficult to implement, expensive to scale, and laborious to adapt by protein engineering.
Thus, there remains a need for improving enzyme-based alkylation.
The technical problem underlying the present invention is thus the provision of a system for improved enzyme-catalyzed alkylation.
The problem is solved by the embodiments as described herein below and as summarized in the appended claims. The present invention provides, in one aspect, a method for alkylating a substrate (briefly referred to as“alkylation method” herein) which comprises the following steps:
a) an alkylation step wherein a S-alkyltransferase transfers an optionally substituted alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound;
b) an alkylation step wherein a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkyiated sulfur- or selenium- containing carrier compound,
and wherein at least a part of the dealkyiated sulfur- or selenium-containing carrier compound yielded in step b) is recycled to step a) to regenerate the alkylated sulfur- or selenium-containing carrier compound.
The steps of the alkylation method in accordance with the invention are schematically illustrated in Figure 31.
Further aspects of the invention relate to kits comprising the employed components and the uses thereof to produce an alkylated substrate utilising the regeneration of spent carrier compound. In accordance with still a further aspect, the invention provides a method for the production of the alkyl carrier compound in its alkylated form (briefly referred to as“carrier production method” herein).
In accordance with the alkylation method defined herein, an alkylated product of interest (i.e. the alkylated substrate) can be efficiently produced. In particular, the use of the alkyltransferase in step b) to transfer the optionally substituted alkyl group from an alkylated carrier compound to the substrate allows the alkylation of the substrate to proceed at a high yield and/or a high specificity for the desired product. The use of the alkylransferase in step a) allows the alkylated carrier to be regenerated, such that the alkylated carrier can be used in amounts which are significantly below stoichiometric amounts, i.e. in amounts which are significantly below the ratio of one mol of alkylated carrier molecule for each mol of optionally substituted alkyl group that is to be transferred to the substrate. Additionally, the design of the alkylation method of the present invention makes alkyl donors available as sources of an afkyl group which are commercially available at lower costs than alkyl carriers disclosed in the literature for enzymatically controlled alkylation reactions, such as SAM. In particular, the alkylation method provided by the invention removes the limitations of the prior art by a simple enzymatic (re-)generating/recycling of the alkyl carrier compound functioning as a co-substrate of an alkyltransferase (e.g (re-)generating S-adenosyl methionine (SAM) from the spent S-adenosyl homocysteine (SAH)), which can be accomplished by a single regenerating enzyme. In the alkylation method of the invention, the co-substrate can be used as a catalytic, i.e. sub-stoichiometric component for the enzyme- catalyzed alkylation. The co-substrate is provided by a carrier compound for the alkyl group, whereas a separate alkyl donor is used as a source of the alkyl group. Alkyltransferases such as methyltransferases can catalyse the dealkylation of a co-substrate/carrier compound such as S-adenosyl methionine (SAM) to form specifically alkylated products and a dealky!ated co-substrate/carrier compound such as S-adenosylhomocysteine (SAH) as side product (Figure 1 ). In the invention, an S-alkyttransferase can realkylate a co- substrate/carrier compound using a cost effective alkyl donor (e.g. remethy!ation of S- adenosylhomocysteine (SAH) to S-adenosyl methionine (SAM) by using methyl iodide or S~ methyl methionine). An enzyme cascade consisting of an S-alkyltransferase (e.g. a halide methyltransferase or S-methionine methyltransferase) and further N-, C-, 0-, S-, or P- alkyltransferase enables the transfer of alkyl groups from an alkyl donor (e.g. methyl iodide or methyl chloride; or S-methyl methionine) to a substrate, using the co-substrate (e.g. S- adenosyl methionine (SAM)) merely as an alkyl transfer catalyst. Such enzyme cascades require only catalytic concentrations of the alkyl carrier compound and use a different source as stoichiometric alkyl donor. The efficient carrier compound regeneration system of the present invention requires only one regenerating enzyme and is compatible with the use of cost effective reagents such as methyl iodide as alkyl donor. The catalytic alkyl carrier compound can be used in more than 500 turnovers in this method (Figure 1 ).
The method of the present invention is broadly applicable and can, inter alia, be applied for b-methyiation of L- and D-a-amino acids, b-methylation of hydroxy acids, b-methylation of alcohols or b-methylation of amines, preferably for b-methylation of L- and D-a-amino acids. In the context of amines and alcohols, b-methylation can also be referred to as 2- methylation. The relevant reaction steps including the involved enzymes of b-methylation or 2-methylation are shown in Figure 46. b-methylation or 2-methylation is relevant because stereoselective introduction of b-methyl groups is difficult using traditional methods of organic synthesis. Accordingly, one advantage of the method of the present invention is a high stereo-selectivity. Furthermore, synthesis of /3-methyl amino acids enables further application of such compounds in medicinal chemistry and/or chemical biology studies. In the following, the alkylation method defined above as a first aspect of the invention will be described in further detail. However, the following definitions also apply to the other aspects of the invention relating to the kit and use thereof, and, as far as the provision of the alkylated sulfur- or selenium-containing carrier compound is concerned, also to the carrier production method further discussed below.
An alkylation step (also referred to herein as alkylation reaction) is a transfer of an alkyl group from one molecule to another molecule. As noted above, the alkyl group which is transferred in the alkylation method or the carrier production method in accordance with the invention is an optionally substituted alkyl group, i.e. an alkyl group which may be non- substituted, or may comprise one or more substituents. An alkyf group is a group which can be derived from an alkane by removal of a hydrogen atom from a carbon atom. An alkylated compound, such as the alkylated carrier compound or the alkylated substrate used in the methods of the present invention, is a compound to which an alkyl group has been transferred {i.e. a compound into which an alkyl group has been introduced, and wherein the alkyl group is covalently bound). As will be appreciated by the skilled reader, this definition does not impose any restrictions with respect to further alkyl groups which may have been present in the compound already prior to the alkylation step. A dealkylated compound, such as the dealkylated carrier compound used in the alkylation method of the present invention, is a compound from which an alkyl group has been transferred (i.e. a compound from which an alkyl group has been removed). Also in this regard, it will be appreciated by the skilled reader that alkyl groups may be present in the dealkylated compound which are not involved in the transfer of an alkyl group. In the context of the present invention, the alkylated carrier compound and the alkylated substrate each comprise the optionally substituted alkyl group which is transferred by the alkyltransferases, while the dealkylated carrier compound and the substrate do not comprise the optionally substituted alkyl group which is transferred by the alkyltransferases.
The alkylation steps which are carried out in the context of the present invention, involve the use of an alky!transferase. An alkyltransferase is an enzyme which catalyzes an alkylation reaction and thus accomplishes a transfer of an (optionally substituted) alkyl group from one compound to another. In the present invention, the alkyltransferase used in step a) and the alkyltransferase used in the carrier production method transfers an optionally substituted alkyl group to a sulfur- or selenium containing carrier compound. The alkyltransferase used in step b) transfers the optionally substituted alkyl group to a substrate. The alkyltransferase used in step a) is also referred to as first alkyltransferase, primary alkyltransferase, regenerating enzyme/regenerating alkyltransferase or enzyme. The alkyltransferase used in step b) is also referred to as second alkyltransferase, or secondary alkyltransferase, further alkyltransferase, or alkylating enzyme.
The alkylation step a) yields an alkylated sulfur- or selenium-containing carrier compound by the transfer of an optionally substituted alkyl group from an alkyl donor. The alkyl donor as used in the context of the present invention acts as a source of an optionally substituted alkyl group. Typically, the alkyl donor is consumed when its optionally substituted alkyl group is transferred to the sulfur- or selenium containing carrier compound, i.e. the alkyl donor is not regenerated. Thus, a sufficient amount of the alkyl donor should be provided for carrying out the alkylation method in accordance with the invention, taking due account of the amount of substrate to be alkylated. Preferably, at least one equivalent of the alkyl donor is provided per equivalent of the optionally substituted alkyl group to be transferred to the substrate.
As noted above, the optionally substituted alkyl group of the alkyl donor is transferred from the alkyl donor to a sulfur- or selenium-containing carrier compound to yield an alkylated sulfur- or selenium-containing carrier compound. For reasons of comprehensibility, the sulfur- or selenium-containing carrier compound which has not yet been alkylated may be briefly referred to as “carrier compound” or as "carrier compound prior to alkylation” herein. Similarly, the alkylated sulfur- or selenenium containing carrier compound may be briefly referred to as“alkylated carrier compound” herein.
The alkylation step b) yields an alkylated substrate as defined herein and a dealkyiated sulfur- or selenium containing carrier compound. The dealkyiated carrier compound can be considered as spent carrier compound. The dealkyiated carrier compound resulting from step b) will generally correspond in its structure to the carrier compound prior to alkylation. In other words, in terms of the compound structure, there are typically no differences between the sulfur- or selenium-containing carrier compound prior to alkylation and the dealkyiated sulfur- or selenium-containing carrier compound. The sulfur- or selenium-containing carrier compound which has been dealkyiated may therefore equally be referred to as “carrier compound”, or as“dealkyiated carrier compound” herein.
From a functional perspective, the alkylated carrier compound can be referred to as an intermediate product in the alkylation method in accordance with the invention which carries an optionally substituted alkyl group received from an alkyl donor to a substrate to be alkylated. In the carrier compound production method in accordance with the invention, the alkylated carrier compound can also be referred to as a product which is capable of carrying an optionally substituted alkyl group received from an alkyl donor.
Since the carrier compound used in step a) may be recovered in the form of the dealkylated carrier compound from step b), the alkylation method in accordance with the invention can be considered as involving a net reaction as the sum of steps a) and b) wherein the substrate is alkylated with the optionally substituted alkyl group provided by the alkyl donor.
At least a part of the dealkylated sulfur- or selenium containing carrier compound yielded in step b) is recycled to step a) in order to regenerate the alkylated sulfur- or selenium containing carrier compound. Typically, the predominant amount (i.e. more than 50 mo!%, based on the total amount of dealkylated carrier compound as 100 mol%) is recycled to step a) to regenerate the alkylated carrier compound For example, the dealkylated sulfur- or selenium containing carrier compound yielded in step b) may be completely recycled to step a). It will be understood that the regenerated alkylated carrier compound resulting from this recycling is available again as a reactant in step b) in the alkylation method in accordance with the invention.
Each sulfur- or selenium containing carrier compound {i.e. each molecule of the carrier compound) used in the alkylation method in accordance with the invention can therefore be subjected to the alkylation step a) multiple times. It will be appreciated that once the alkylation step a) has been accomplished, the resulting alkylated sulfur- or selenium- containing carrier compound undergoes step b) to allow its optionally substituted alkyl group to be transferred to the substrate and to complete a cycle by yielding the dealkylated carrier compound which can then again be subjected to the alkylation step a).
Typically, each sulfur- or selenium containing carrier compound molecule used in the alkylation method in accordance with the invention is subjected to the alkylation step a) on the average at least 2 times, preferably at least 5 times, more preferably at least 10 times, even more preferably at least 50 times, still more preferably at least 100 times, and most preferably at least 500 times. Due to the subsequent transfer of the optionally substituted alkyl from the alkylated carrier compound to the substrate in step b), this average number of alkylation steps can be conveniently calculated from the yield of the alkylated substrate provided by the alkylation method and the amount of carrier compound used. This means that, in a preferred case, the alkyltransferase of step b) may transfer at least 5, more preferably at least 10, even more preferably at least 50, still more preferably at least 100, and most preferably at least 500 alkyl groups to substrate molecules to be alkylated per sulfur- or selenium-containing carrier compound molecule used.
For this reason, the carrier compound can be used in amounts in the alkylation method in accordance with the invention such that less than one molecule of the carrier compound is provided for each optionally substituted alkyl group that is to be transferred to the substrate.
Since the carrier compound can undergo multiple alkylation reactions, it is possible to carry out step a) as a first alkylation step, followed by step b) as a second alkylation step, or to carry out step b) as a first alkylation step, followed by step a) as a second alkylation step.
In order to carry out step a) as a first alkylation step, the carrier compound prior to alkylation may be used as a starting compound together with the alkyl donor and the S-alkyltransferase of step a). Thus, the alkylation of the carrier compound to provide the alkylated carrier would proceed as the first step in the reaction system containing these components, and the alkylated carrier compound can subsequently be used in step b), advantageously in the same reaction system.
In order to carry out step b) as a first alkylation step, an alkylated carrier compound can be obtained or can be preliminarily synthesized in a separate procedure, and can then be used as a starting compound together with the N-. C-, 0-, S-, or P-alkyltransferase of step b) and the substrate to be alkylated. Thus, the alkylation of the substrate and the dealkylation of the alkylated carrier would proceed as the first step in the reaction system containing these components, and the dealkylated carrier can subsequently be recycled to a step a) to regenerate the alkylated carrier compound. Preferably, the alkylation of step a) is the first alkylation step, followed by step b) as the second alkylation step.
fn line with the above, the alkylation method in accordance with the invention may also be referred to as a method for alkylating a substrate which comprises the following steps::
a) an alkylation step, wherein a S-alkyltransferase transfers an optionally substituted alkyl group from an alky! donor to a sulfur- or selenium-containing carrier yielding an alkylated sulfur- or selenium-containing carrier compound;
b) an alkylation step, wherein a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier to the substrate yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound, and
c) a regeneration step, wherein the S-specific alkyltransferase transfers an optionally substituted alkyl group from an alkyl donor to the dealkylated sulfur- or selenium-containing carrier obtained in step b) to regenerate an alkylated sulfur- or selenium-containing carrier compound,
wherein each sulfur- or selenium-containing carrier compound molecule used in the method is subjected to steps b) and c) on the average at least two times.
As will be appreciated, the definitions and preferred definitions of the optionally substituted alkyl group, the alkyl donor, the S-alky!transferase, the N-, C-, 0-, or P-alkyltransferase, the sulfur- or selenium-containing carrier compound and the substrate provided herein for the alkylation method in accordance with the invention continue to apply for this preferred embodiment, Moreover, the alkyl donor in of the regeneration step c) preferably has the same structure as the alkyl donor of step a). Preferably, the alkyl donor in recycling step c) does not relate to the same alkyl donor molecule. Preferably the alkyl donor in recycling step c) relates to a newly added alkyl donor molecule. Alkylation step a) is preferably the first alkylation step that is carried out prior to steps b) and c).
Preferably, each sulfur- or selenium-containing carrier compound molecule used in the preferred method is subjected to steps b) and c) on the average at least 5 times, more preferably at least 10 times, even more preferably at least 50 times, still more preferably at least 100 times and most preferably at least 500 times. This means that, in a preferred case, the alkyltransferase of step b) may transfer at least 5, more preferably at least 10, even more preferably at least 50, still more preferably at least 100, and most preferably at least 500 alkyf groups to substrate molecules to be alkylated per sulfur- or selenium-containing carrier compound molecule used.
The alkylation method and the carrier production method in accordance with the invention can be carried out in vitro or in vivo. The methods can be conveniently carried out at or around room temperature, e.g. in a temperature range of 10 to 40 °C, more preferably 15 to 30 X. If the method is carried out in vitro, it is preferably carried out in a cell free-system or alternatively in a buffer.
A celf-free system may be an extract from a cell, which expresses the aikyttransferases used in steps a) and b). The cell extract can be obtained by lysis of the cell (cell-free lysate). If the method is carried out in a buffer, the alkyltransferases used in steps a) and b) can be obtained from a cell, for example by purification, and added to the buffer. A typical buffer is a sodium phosphate buffer, such as a 100 mM sodium phosphate buffer with a pH of 8,0.
In a preferred embodiment, the alkylation method in accordance with the invention is carried out (as an in vitro method) by combining the alkyl donor, the sulfur- or selenium-containing carrier compound, the S-alkyltransferase used in step a), the N-, C-, 0-, S-, or P- alkyltransferase used in step b) and the substrate to be alkylated in a single reaction vessel, before allowing the reaction to proceed.
If the method is carried out in vivo, it is preferably carried out in a host cell. The host cel! is preferably a non-human or non-animal cell, more preferably a bacterial cell, plant cell or fungal cell. Particularly preferred bacterial cells are E.coli and C. Glutamicum. The alkyltransferases of step a) and b) may be expressed in the same or different host cells. The skilled person is familiar with suitable host cell and expression systems. For example, a gene encoding an alkyltransferase of the present invention may be cloned into a vector under control of a promoter such as an inducible promoter. After induction of the promoter, the gene is expressed and the method can be carried out.
It is preferred that the method is carried out in the absence of S-adenosyl adenosine nucleosidase (EC 3.2.2.9), when S-adenosyl homocysteine is used as a carrier compound. If the method is carried out in vivo, the host cell is preferably S-adenosyl adenosine nucleosidase (EC 3.2.2.9) deficient.
If the method is used for labeling a substrate in vivo, the method may be carried out in the absence of the methionine adenosyl transferase (MAT) to avoid a competition with unlabelled S-adenosyl methionine (SAM), which is generated by native S-adenosyl methionine (SAM) production in the host cell. Thus, the host cell used may be methionine adenosyl transferase (MAT) deficient. It may be further required express the S-adenosyl transporter in the host cell, which is methionine adenosyl transferase (MAT) deficient, to ensure viability of the host cell. It may also be required to overexpress the nucleoside transporter and NupC and/or NupG to increase the intracellular adenosine concentration. It may further be required to overexpress the genes for MetA, MetB and MetC to enable production of homocysteine. Finally, it may also be required to overexpress adenosy!homocysteine hydrolase {EC 3.3.1.1) to enable production of SAH from homocysteine and adenosine.
The method for alkylating a substrate according to the present invention does not require amounts of the sulfur- or selenium-containing carrier compound which are at least stoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate, i.e, it is not necessary to provide an amount corresponding to at least one mol or more of the sulfur- or selenium-containing carrier compound for each moi of optionally substituted alkyl group that is to be transferred to the substrate. This is advantageous as sulfur- or selenium-containing carrier compound such as S-adendosyi methionine (SAM) can be very expensive. Preferably, the sulfur- or selenium-containing carrier compound is present in an amount which is substoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate, i.e. an amount corresponding to less then one mol of the sulfur- or selenium-containing carrier compound for each mol of the optionally substituted alkyl group that is to be transferred to the substrate.. The alkyl donor used in the present invention is preferably inexpensive. The alkyl donor is preferably used in the alkylation method and the carrier production method in an amount which is at least stoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate, i.e. an amount corresponding to at least one mol of the alkyl donor for each mot of the optionally substituted alkyl group that is to be transferred to the substrate.. It will be appreciated that this includes the possibility that the alkyl donor is used in an amount which is more than stoichiometric to the amount of alkyl group to be transferred to the substrate,.
It will be understood that the reference herein to the (molar) amount of the alkyl group that is to be transferred to the substrate relates to the alkyl groups that can actually be bound to/accepted by the substrate, and which corresponds to the (molar) amount of substrate to be alkylated by the alkylation method in accordance with the invention, where applicable multiplied by the number of alkyl groups to be transferred to each substrate molecule in cases where multiple alky! groups can be transferred to a single substrate molecule. Sulfur- or selenium-containing carrier compc
In step a) of the method for alkylating a substrate in accordance with the present invention, a S-atkyltransferase transfers an optionally substituted alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium- containing carrier compound; and further a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound.
The sulfur- or selenium-containing carrier compound (i.e. a carrier compound containing a sulfur or selenium atom) can also be referred to as a sulfur- or selenium-containing cosubstrate or co-factor. The sulfur- or selenium-containing carrier compound is used according to the present invention as a carrier for carrying an optionally substituted alkyl group from an alkyl donor to a substrate. In accordance with the alkylation method, the sulfur- or selenium-containing carrier compound is alkylated in step a) with an optionally substituted alkyl group yielding an alkylated sulfur- or selenium-containing carrier compound and the alkylated sulfur- or selenium-containing carrier compound is dealkylated in step b) yielding a dealkylated sulfur- or selenium-containing carrier compound (i.e. yielding again the sulfur- or selenium-containing carrier compound used in step a)). The optionally substituted alkyl group of the alkylated sulfur- or selenium-containing carrier compound is generally covalently bound to the sulfur or selenium atom of the carrier compound. Typically, the sulfur- or selenium-containing carrier compound contains one sulur- or selenium atom, preferably one sulfur atom, and is able to carry one optionally substituted alkyl group in its alkylated form.
As described above, the alkylated sulfur- or selenium containing carrier compound is the sulfur- or selenium containing carrier compound comprising the optionally substituted alkyl group, or, in other words, the sulfur- or selenium containing carrier compound to which the optionally substituted alkyl group has been transferred.
Preferably, the sulfur-containing carrier compound prior to alkylation (and after dealkylation) comprises a thioether moiety. A thioether moiety contains a sulfur atom which is connected to two carbon atoms. The thioether moiety may be represented by the following structural unit:
Figure imgf000013_0001
It is noted that, in the preferred case where the sulfur-containing carrier compound comprises a thioether moiety, the optionally substituted alkyl group of the alkylated sulfur-containing carrier compound is covalently bound to the sulfur atom of the thioether moiety. Thus, the alkylated sulfur-containing carrier compound may represent a sulfonium ion.
The sulfur-containing carrier compound prior to alkylation (and after dealkylation) is preferably represented by the following formula (la):
Figure imgf000014_0001
wherein,
R1 is selected from the group consisting of a carboxy group, hydrogen, a triazole and an ester group;
R2 is selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy group;
R3 and R4 are independently selected from hydrogen and a hydroxy group, and are preferably a preferably a hydroxy group; and
B1 is an optionally substituted purine or thienoadenine.
The sulfur-containing carrier compound represented by formula (la) is preferably in L- configuration, such that the sulfur-containing carrier compound prior to alkylation (and after dealkylation) may be represented by the following formula (laa):
Figure imgf000014_0002
wherein R1, R2, R3, R4 and B1 are as defined above.
As will be understood by the skilled reader, the ester group represents a carboxylate ester group, wherein the hydroxy group of the carboxylic acid is replaced by an alkoxy group The alkoxy group preferably contains 1 to 9 carbon atoms. Examples of the alkoxy group include methoxy, ethoxy, propoxy, butoxy, phenoxy and benzyloxy. A primary amino group represents an amino group, wherein one of the three substituents bound to the nitrogen atom is an organic group other than hydrogen A secondary amino group represents an amino group wherein two of the three substituents bound to the nitrogen atom are organic groups other than hydrogen A tertiary amino group represents an amino wherein the three substituents bound to the nitrogen atom are organic groups other than hydrogen. The organic group is preferably an aliphatic hydrocarbon group, more preferably an alkyl group, having 1 to 5 carbon atoms. Suitable examples of the secondary amino group include methylamino, ethylamino, propylamino, allylamino and butylamino, preferably methylamino. Suitable examples of the tertiary amino group include dimethylamino, diethylamino, dipropylamino, and dibutylamino, preferably dimethylamino.
The optionally substituted purine may be substituted by one or more, such as one, two or three, substituents. Exemplary substituents for the optionally substituted purine are selected from the group consisting of an alky! group having 1 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, an oxo group, a halogen atom, a hydroxy group, an amino group, a secondary amino group, and a tertiary amino group. Examples of the optionally substituted purine include purine, 6-hydroxypurine, adenine, guanine, N6-alkyl adenine (alkyl = methyl, ethyl, propyl, benzyl, isopropyl and 2-naphtylmethyl), 2- fluoroadenine, and 2-ch!oroadenine.
In formulas (la) and (iaa), R1 is preferably selected from the group consisting of a carboxy group, hydrogen and a ester group; R2 is preferably selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group; R3 and R4 are preferably a hydroxy group; and B1 is preferably an optionally substituted adenine.
The optionally substituted adenine may be substituted by one or more, such as one, two or three, substituents. Exemplary substituents for the optionally substituted adenine are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, and a halogen atom.
The sulfur-containing carrier compound prior to alkylation is more preferably selected from the group consisting of S-adenosyl homocysteine (SAH), 2-fluoroadenosyi homocysteine, and S-(5'-adenosyl)-3-thiopropylamine, Nd-substituted S-adenosyl homocysteine, S- adenosyl N-acetylhomocysteine, S-adenosyl N,N,-dimethylhomocysteine, S-adenosyl homocysteine methylester, S-adenosyl homocysteine ethylester and S-adenosyl homocysteine phenylester.
The N6-substituted S-adenosyl homocysteine is an S-adenosyl homocysteine which is substituted at the Nd-position with an aliphatic or aromatic hydrocarbon group having 1 to 12 carbon atoms. Examples of N6-substituted S-adenosyl homocysteine include NS-methyt S- adenosyl homocysteine, N6-ethyl S-adenosyl homocysteine, N6-propyl S-adenosyl homocysteine, Nd-benzyl S-adenosyl homocysteine, N6-isopropyl S-adenosyl homocysteine and N6-2-naphtylmethy! S-adenosyl homocysteine, preferably N6-methyl S-adenosyl homocysteine, N6-ethyl S-adenosyl homocysteine, N6-propyl S-adenosyl homocysteine, and N6-isopropyl S-adenosyl homocysteine.
The alkylated sulfur-containing carrier compound is preferably a methylated sulfur-containing carrier compound. Examples of the methylated sulfur-containing carrier compound include S- adenosy! methionine (SAM), 2-fluoroadenosyl methionine, and S-methyl-S-(5'-adenosyl)-3- thiopropylamine, N6-substituted S-adenosyl methionine, S-adenosyl N-acetylmethionine, S- adenosyl N,N,-dimethylmethionine, S-adenosyl methionine methylester, S-adenosyl methionine ethylester and S-adenosyl methionine phenylester.
The sulfur-containing carrier compound prior to alkylation (and after dealkylation) is most preferably S-adenosyl homocysteine (SAH). The alkylated sulfur-containing compound yielded in step b) is most preferably S-adenosyl methionine (SAM).
Alternatively, the sulfur-containing carrier compound may be represented by the following formula (lb):
Figure imgf000016_0001
wherein R5 is selected from the group consisting of a methyl group which may be substituted, an ethyl group which may be substituted and a propyl group which may be substituted.
Exemplary substituents of R5 are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 2 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, an oxo group, a halogen atom, a hydroxy group, an amino group, a secondary amino group, and a tertiary amino group. The substituents of R5 are preferably a halogen atom, more preferably a fluorine atom.
Suitable examples of the sulfur-containing carrier compound represented by formula (lb) include methionine, monofluoromethyl homocysteine, difluoromethyl homocysteine and trifluoromethyl homocysteine. Examples of the alkylated sulfur-containing carrier compound of formula (lb) include S-methyl methionine, monofluoromethyl methionine, difluoromethyl methionine and trifluoromethyl methionine. If a selenium-containing carrier compound is used, the selenium-containing carrier compound prior to alkylation (and after dealkylation) preferably comprises a selenoether moiety. A selenoether moiety contains a selenium atom which is connected to two carbon atoms. The selenoether moiety may be represented by the following structural unit:
Figure imgf000017_0001
It is noted that, in the preferred case where the selenium-containing carrier compound comprises a selenoether moiety, the optionally substituted alkyl group of the alkylated selenium-containing carrier compound is covalently bound to the selenium atom of the selenoether moiety. Thus, the alkylated selenium-containing carrier compound may represent an ion analogous to a sulfonium ion.
The selenium-containing carrier compound prior to alkylation (and after dealkylation) is preferably represented by the following formula (lc):
Figure imgf000017_0002
wherein,
R6 is selected from the group consisting of a carboxy group, a hydrogen, a triazole, and an ester;
R7 is selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy;
R8 and R9 are independently selected from hydrogen and a hydroxy group, preferably a hydroxy group; and B2 is an optionally substituted purine or thienoadenine.
The selenium-containing carrier compound represented by formula (lc) is preferably in L- configuration such that the selenium-containing carrier compound prior to alkylation may be represented by the following formula (lea):
Figure imgf000018_0001
wherein R6, R7, R8, R9 and B2 are as defined above.
The ester group, the primary amino group, the secondary amino group, the tertiary amino group, and the optionally substituted purine are as defined above for the sulfur-containing carrier compound of formulas (la) and (laa).
In formulas (lc) and (lea), R6 is preferably selected from the group consisting of a carboxy group, hydrogen and a ester group; R7 is preferably selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group; R8 and R9 are preferably a hydroxy group; and B2 is preferably an optionally substituted adenine.
The optionally substituted adenine may be substituted by one or more, such as one, two or three, substituents. Exemplary substituents for the optionally substituted adenine are selected from the group consisting of an alkyl group having 1 to 5 carbon atoms, an aromatic hydrocarbon group having 5 to 12 carbon atoms, an aralkyl group having 6 to 12 carbon atoms, and a halogen atom.
The selenium-containing carrier compound prior to alkylation is more preferably selected from the group consisting of Se-adenosyl selenohomocysteine, thienoadenosyl selenohomocysteine, Se-(2-fluoroadenosyI) selenohomocysteine, Se-(5'-adenosyl)-3- selenopropylamine, N6-substituted Se-adenosyl selenohomocysteine, Se-adenosyl N-acetyl selenohomocysteine, Se-adenosyl N,N, -dimethyl selenohomocysteine, Se-adenosyl selenohomocysteine methylester, Se-adenosyl selenohomocysteine ethylester and Se- adenosyl selenohomocysteine phenylester.
The N6-substituted Se-adenosyl selenohomocysteine is an Se-adenosyl selenohomocysteine which is substituted at the N6-position with an aliphatic or aromatic hydrocarbon group having 1 to 12 carbon atoms. Examples of N6-substitufed Se-adenosyl homocysteine include N6-methyl Se-adenosyl homocysteine, N6-ethyl Se-adenosyl homocysteine, N6-propyl Se-adenosyl homocysteine, N6-benzyl Se-adenosyl homocysteine, N6-isopropyl Se-adenosyl homocysteine and N6-2-naphty!methyl Se-adenosyl homocysteine, preferably N6-methyl Se-adenosyl homocysteine, N6-ethyl Se-adenosyl homocysteine, N6-propyl Se-adenosyl homocysteine, and N6-isopropyl Se-adenosyl homocysteine.
Examples of the alkylated selenium-containing carrier compound include Se-adenosyl selenomethionine, thienoadenosyl selenomethionine, Se-(2-f!uoroadenosyl) selenomethionine, Se-methyl-Se-(5'-adenosyl)-3-selenopropylamine, N6-substituted Se- adenosyl selenomethionine, Se-adenosyl N-acetyl selenomethionine, Se-adenosyl N,N,- dimethyl selenomethionine, Se-adenosyl selenomethionine methylester, Se-adenosyl selenomethionine ethylester and Se-adenosyl selenomethionine phenylester.
Generally, a preference is given in thecontext of the present invention to the use of a sulfur containing carrier compound compared to a selenium containing carrier coumpound. Most preferably, the carrier compound prior to alkylation (and after dealkylation) is S-adenosyl homocysteine (SAH). The alkylated sulfur-containing compound yielded in step b) is most preferably S-adenosyl methionine (SAM).
In the alkylation method of the present invention, the sulfur- or selenium-containing carrier compound may be used in a substoichiometric amount (in particular a catalytic amount) compared to the amount of the optionally substituted alkyl group to be transferred to the substrate. Preferably, the sulfur- or selenium-containing carrier compound is present in an amount of 0.01 mol% to 20 mol%, more preferably 0.05 moI% to 10 moi%, and still more preferably 0.1 mol% to 5 mol%, for each mole of the optionally substituted alkyl group to be transferred to the substrate.
Alkyl transferases
In the methods according to the present invention alkyltransferases are used to transfer the optionally substituted alkyl group to an atom in a carrier compound or a substrate. Depending on the atom to which the optionally substituted alkyl group is transferred, the alkyltransferase is termed N-, C-, 0-, 5-, or P-alkyltransferase. An S-alkyltransferase may also be referred to as S-specific alkyftransferase. An S- alkyltransferase or S-spedfic alkyltransferase is capable of transferring an optionally substituted alkyl group to a sulfur atom, which may be comprised in a substrate or a carrier compound and which is preferably a nucleophilic sulfur atom. An S-alkyltransferase or S- specific alkyltransferase may also be capable of transferring an optionally substituted alkyl group to a selenium atom, which may be comprised in a substrate or the carrier compound and which is preferably a nucleophilic selenium atom. An S-aikyltransferase may be used in step a) and/or b). The alkyltransferase or S-alkyltransferase used in step b) typically differs from the alkyltransferase or S-alkyltransferase used in step a). This means that the alkyltransferase or S-alkyltransferase used in step b) is not the same as the alkyltransferase or S-alkyltransferase used in step a).
An N-alkyltransferase may also be referred to as N-specific alkyltransferase. An N- alkyltransferase is capable of transferring an optionally substituted alkyl group to a nitrogen atom comprised in a substrate. A C-atkyltransferase may also be referred to as C-specific alkyltransferase. A C-alkyltransferase or C-specific alkyltransferase is capable of transferring an optionally substituted alkyl group to a carbon atom comprised in a substrate. An O- alkyltransferase may also be referred to as Q-specific alkyltransferase. An O-alkyltransferase or O-specific alkyltransferase is capable of transferring an optionally substituted alkyl group to an oxygen atom comprised in a substrate. A P-alkyltransferase may also be referred to as P-specific alkyltransferase. A P-alkyltransferase or P-specific alkyltransferase is capable of transferring an optionally substituted alkyl group to a phosphorus atom comprised in a substrate. Preferably, the N-, C-, 0-, S- or P-alkyltransferase used in step b) does not transfer an optionally substituted alkyl group to a carrier compound.
In a particularly preferred embodiment the alkyltransferase of step a) and/or the alkyltransferase of step b) is a methyltransferase. The alkylation catalyzed by the alkyltransferases disclosed herein may be considered as being reversible or irreversible for practical purposes. According to the present invention, the S-aikyftransferases or S-specific alkyltransferases may transfer the optionally substituted alkyl group reversibly. Without wishing to be bound by theory, it is assumed that the low pKa of S-protonated thioethers leads to a high alkyl transfer potential of the sulfonium group of alkylated thioethers. On the other hand, most nucleophiles subjected to enzyme catalyzed alkylation form much weaker acids, making alkyl transfers from alkylated thioethers essentially irreversible. The alkylation catalyzed by the N-, C-, 0-, and P-alkyltransferases preferably transfer the optionally substituted alkyl group irreversibly. The alkyltransferase used in step a) preferably transfers the optionally substituted alkyl group reversibly to the sulfur- or selenium containing carrier compound, The alkyltransferase used in step b) preferably transfers the optionally substituted alkyl group irreversibly to the substrate.
The alkyltransferase used in step a) and/or b) may be an S-adenosyl methionine (SAM) dependent alkyltransferase. An S-adenosyl methionine (SAM) dependent alkyltransferase transfers an optionally substituted alkyl group in the presence of S-adenosyl methionine.
An S-adenosyl methionine (SAM) dependent alkyltransferase may transfer an optionally substituted alkyl group to a carrier compound, preferably a sulfur-containing carrier compound, more preferably S-adenosyl homocysteine (SAH). This transfer is preferably carried out by the S-adenosyl methionine (SAM) dependent alkyltransferase used in step a).
An S-adenosyl methionine (SAM) dependent alkyltransferase may also transfer an optionally substituted alkyl group to a substrate in the presence of S-adenosyl methionine. This transfer is preferably carried out by the S-adenosyl methionine (SAM) dependent alkyltransferase used in step b).
For an efficient recycling of the dealkylated sulfur- or selenium-containing carrier compound obtained in step b), it is important that the dealkylated sulfur- or selenium-containing carrier compound obtained in step b) is not degraded. S-adenosyl homocysteine may be degraded by hydrolysis to adenine and ribosyl homocysteine (see Figure 3 and figure 8). To minimize the degradation of S-adenosyl homocysteine, the method is preferably carried out in the absence of S-adenosyl homocysteine (SAH) nucleosidase (EC 3,2.2 9),
The absence of the S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) is particularly relevant if S-adenosyl homocysteine (SAH) or Se-adenosyl selenohomocysteine is used as sulfur- or selenium-containing carrier compound.
In the context of the present invention, the term "in the absence of the S-adenosyl homocysteine (SAH) nucleosidase” means that the activity of the S-adenosyl homocysteine (SAH) nucleosidase is less than 0.05% mol units per mg alkyltransferase, preferably 0.025% mol units per mg alkyltransferase, more preferably less than 0,013% mol units per g alkyltransferase; and/or that the activity of the S-adenosyl homocysteine (SAH) nucleosidase may less than 1 pmo!/min per mg alkyltransferase, preferably 0.5 pmol/min per mg alkyltransferase, more preferably less than 0.25 pmol/min per mg alkyltransferase.
The absence of the adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) may be achieved additional purification of the alkyltransferases used in steps a) and b), for example by an additional chromatographic step, such as a size exclusion chromatography. An alkyltransferase, which was additionally purified, is also referred to as pre-purified alkyltransferase or re-purified enzyme. Alternatively, the absence of the adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) may be achieved by using an S-adenosyl homocysteine (SAH) nucleosidase (EC 3, 2.2.9) deficient host cell for expressing the alkyltransferases. To obtain an S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) host cell, gene encoding the alkyltransferases S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) is deleted prior to expression of the alkyltransferase.
The alkyltransferase of the present invention may be obtained by expression in a cell. Expression may be native or recombinant. Native expression refers to expression of the alkyltransferase in a cell from an organism, from which the alkyltransferase originates. Recombinant expression refers to expression of the alkyltransferase in a host cell. The host cell is preferably not from the organism from which the alkyltransferase originates. Any suitable host cell may be used. Preferably the host cell is a bacterial cell, plant cell, fungal cell or insect cell. The host cell is preferably a bacterial cell, more preferably E, coli. Preferably, the host cell is S- adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) deficient. The skilled person is familiar with the host cells and expression systems, For example, a gene encoding an alkyltransferase of the present invention may be cloned into a vector under control of a promoter, preferably an inducible promoter. After transfer of the vector into a host cell, expression of the alkyltransferase in the host cell may be initiated by the promoter. After expression, the alkyltransferase may be purified from the host cell. Suitable purification schemes are known to the skilled person. For example, the cells may be lysed and centrifuged to remove cell debris. The alkyltransferase can then be purified from the obtained supernatant, for example by affinity chromatography such as Ni“ affinity chromatography. Additional purification steps, such as ion exchange chromatography or size exclusion chromatography may be used to further purify the alkyltransferase. Additional purification steps may be used to remove contaminating enzymes, such as the S- adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2,9). Alkyltransferase used in step a)
The alkyltransferase used in step a) is an S-alkyltransferase, preferably an S- methyltransferase.
In a preferred embodiment, the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT; EC 2.1.1 ,165), thioether methyltransferases, N-methyl proline methyltransferase, N-dimethyi glycine methyltransferase and N-dimethyl beta-aianine N-methyltransferase. The thioether methyltransferase is preferably methionine S-methyltransferase (MSMT; EC 2.1 1.12).
The N-specific methyltransferases selected from the group consisting of N-methyl proline methyltransferase, N-dimethyl glycine methyltransferase and N-dimethyl beta-alanine N- methyltransferase can also accept sulfonium compounds and can transfer an alkyl group to a sulfur or selenium atom, e.g. comprised in a carrier compound such as S-adenosyl homocysteine (SAH). Accordingly, N-methyl proline methyltransferase, N-dimethyl glycine methyltransferase and N-dimethyl beta-alanine N-methyltransferase are capable of alkylating S-adenosyl homocysteine (SAH) and may, thus, be an S-alkyltransferase used in step a) as defined herein.
The above alkyltransferases are preferably S-adenosyl methionine (SAM) dependent methyltransferases.
In a particularly preferred embodiment, the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT) and the methionine S- methyltransferase (MSMT). The halide methyl transferases (HMT) and the methionine S- methyltransferase (MSMT) are independently preferably S-adenosyl methionine (SAM) dependent. The halide methyl transferase (HMT) is preferably used as alkyltransferase in step a), when the alkyl donor is methyl halide such as methyl iodide or methyl chloride. The halide methyl transferase (HMT) preferably originates from bacteria, plants, fungi or archaea, more preferably the halide methyl transferase (SEQ ID NO: 1 ) originates from an acidobacterium, even more preferably from chloracidobacterium thermophilum. In addition, the halide methyl transferase (HMT) can originate from fungi, bacteria, marine algae, diatoms or halophytic plants. The term “originates from” in the present invention means that the enzyme, in particular the alkyltransferase, is encoded by the organism indicated as origin. The term does not indicate the organism, in which the enzyme, in particular the alkyltransferase, was expressed. Preferably, the alkyltransferase is expressed recombinantly in a host cell, which differs from the organism indicated as origin.
The methionine S-methyltransferase (MSMT) is preferably used as alkyltransferase in step a), when the alkyl donor is S-methyl methionine. The methionine S-methyltransferase (MSMT) preferably originates from bacteria, plants, fungi or archaea, more preferably the methionine S-methyltransferase (MSMT) originates from a rhodobacter, even more preferably from Rhodobacter sp. JA431
Thioether methyltransferases are preferably used as alkyltransferase in step a), when the alkyl donor is a S-methylated cyclic thioether such as S-methyl tetrahydrothiophen, S-methyl thietane, and S-methyl ethylene sulphide,
N-methyi proline methyltransferases, N-dimethyi glycine methyltransferases and N-dimethyl beta-alanine N-methyltransferases may be used as alkyltransferase in step a), when the alkyl donor is S-methyl 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid, and S- methyl 3-(methylthio)propionic acid, respectively.
Thioether methyltransferases (also known as indolethylamine N-methyltransferase or amine N-methyitransferase - which better describe the physiological function of this enzyme) can methylate dimethylsulfides and other small thioethers using SAM as methyl donor to form methyl sulfonium ions. Based on the observations with halide methyl transferase (HMT) disclosed herein, it can be reasonably expected that specific methyl sulfonium cations (including trialkyl sulfoxonium cations) can also transfer a methyl group (or other small alkyl groups) to S-adenosyl homocystein (SAH). By analogy, it is reasonable to expect that similar enzymes such as phenylethanolamine N-methyltransferase can catalyze methylation of S- adenosyl homocysteine (SAH) using sulfonium cations such as S-methyl dimethylsulfide as methyl donor. Thioether methyltransferases preferably originate from a vertebrate such as a human or an animal. There are no known bacterial or homologs of these enzymes.
In a further embodiment, the alkyltransferase used in step a) is a carboxylmethyltransferase. Carboxylmethyltransferase are preferably used as alkyltransferase in step a), when the alkyl donor is an oxygen-based alkyl donor of formula (II) as described below, preferably dimethyl carbonate. Dimethylcarbonate is used as an versatile, cost-effective, non-toxic, biodegradable methyldonor reagent, i.e. a green alternative to dimethyl sulfate or methyl halides and phosgene, which could be used for methylation. Based on the observations with the halide methyltransferase as alkyltransferase used in step a) and the methionine S- methyltransferase as alkyltransferase used in step a), it can be expected that carboxymethyltransferases are also suitable methyltransferase for step a). In this system dimethylcarbonate (or a derivative thereof) reacts with S-adenosyl homocysteine (SAH) to produce S-adenosyl methionine (SAM), methanol and C02. The key advantages of this system are i) the synthetic methy!donor produces only volatile side products; ii) dimethyl carbonate is less toxic and less expensive than methyliodide.
The carboxymethyltransferase may originate from bacteria, plants, fungi or archaea, more preferably from plants. More preferably, the carboxymethyltransferase in this embodiment is selected from the group consisting of an indole-3-acetic acid carboxyl methyltransferase, Trigonelline synthase, Gibberellic acid methyltransferase, salicylic acid carboxyl methyltransferase, Jasmonic acid carboxyl methyltransferase, and Caffeine synthase. The indole-3-acetic acid carboxyl methyltransferase preferably originates from Arabidopsis thaliana, the Trigonelline synthase preferably originates from Trigonella foenum-graceum, the Gibberellic acid methyltransferase preferably originates from Arabidopsis thaliana, the salicylic acid carboxyl methyltransferase preferably originates from Petunia x hybrid, the Jasmonic acid carboxyl methyltransferase preferably originates from Arabidopsis thaliana, and the Caffeine synthase preferably originates from Coffea Arabica.
Alkyltransferase used in step b)
The alkyltransferase of step b) is an N-, C-, 0-, S- or P-alky!transferase, which preferably differs from the alkyltransferase used in step a). Preferably, the alkyltransferase of step b) is an N-, C-, 0-, or P-alkyltransferase, more preferably an N-, C-, or O-afkyltransferase. The alkyltransferase used in step b) is preferably a N-, C-, 0-, S- or P-methyltransferase, and thus it is further preferred that the alkyltransferase used in step b) is an N-, C-, 0-, or P- methyltransferase, and most preferred that it is an N-, C-, or O-methyltransferase.
In one embodiment, the alkyltransferase used in step b) is selected from the alkyltransferases disclosed in WO 2013/029075 A, in particular a methyltransferase selected from the group consisting of EC 2.1.1.1 nicotinamide N-methyl transferase, EC 2.1.1.2 Guanidinoacetate N-methyl transferase, EC 2.1.1.4 acetylserotonin O-methyl transferase, EC 2.1.1.6 catechol O-methyl transferase, EC 2.1.1.7 nlcotinate N-methyltransferase, EC 2.1.1.8 histamine N- methyltransferase, EC 2, 1.1.9 thiol S-methyltransferase, EC 2.1.1.12 Methionine S-methyltransferase, EC 2.1.1.15 faty acid O-methyltransferase, EC 2.1.1.16 methylene fatty acid cylphospholipid synthase, EC 2.1.1.17 phosphatidylethanolamine N- methyltransferase, EC EC 2.1.1.20 glycine N methyltransferase, EC 2.1 1.22 carnosine N- methyl transferase, EC 2.1.1.25 phenol 0- methyltransferase, EC 2.1 1.26 lodophenol O- methyl transferase, EC 2.1.1.27 tyramine N- methyltransferase, EC 2.1.1.28 phenylethanolamine N-methyltransferase, EC 2.1. 1 :38 O-Demethylpuromycin O-methyl transferase, EC 2.1.1.39 inositol 3-methyl transferase, EC 2.1.1 ,40 inositol 1 -methyl transferase, EC 2,1.1.41 sterol 24-C-methyltransferase, EC 2.1.1.42 luteolin O- methyltransferase, EC 2.1.1.44 Dimethylhistidin N-Methyltransfe rase, EC 2.1.1.46 isoflavone 4'-0- methyltransferase, EC 2.1 .1.47 !ndolepyruvat C-methyltransferase, EC 2.1.1.49 amine N- methyltransferase, EC 2.1.1.50 Loganat O-methyl transferase, EC 2.1.1.53 putrescine N-methyltransferase, EC 2.1.1.59 [cytochrome cRysin N- methyltransferase, EC 2.1.1.64 3-Demethylubiquinon-9 3-0-methyltransferase, EC 2.1.1.65 Licodion 2’-0-methyl transferase, EC 2.1.1.67 thiopurine S-methyltransferase, EC 2.1. 1.68 Caffeat O-methyl transferase, EC 2.1.1.69 5- Hydroxyfurancoumarin 5-O-methyltransferase, EC 2.1.1.70 8-Hydroxyfurancoumarin 8-0- methyltransferase, EC 2.1.1.71 phosphatidyl-N- methylethanolamine, N-methyl transferase, EC 2.1.1.75 apigenin 4'- O-methyltransferase, EC 2.1.1.76 quercetin 3-0-methyl transferase, EC 2.1.1.78 !soorientin 3'-0-methyl transferase, EC 2.1.1.79 cyclopropane fatty acyl-phospholipid synthase, EC 2.1.1.82 3- methyl quercetin 7- O-methyl transferase, EC 2.1.1.83 3,7-Dimethylquercetin 4'-0- methyltransferase, EC 2.1.1.84 Methylquercetagetin 6-O-methy! transferase, EC 2.1.1.87 pyridine N-methyltransferase, EC 2.1.1.88 8-Hydroxyquercetin 8-O-methyl transferase, EC 2.1.1.89 Tetrahydrocolumbamin 2-0- methyltransferase, EC 2.1.1.91 Isobutyraldoxim O- m ethyl transferase, EC 2.1.1.94 tabersonine 16-O-methyitransferase, EC 2.1.1.95 tocopherol O-methyltransferase, EC 2.1.1.96 thioether S- ethyl transferase, EC 2.1.1.97 3- Hydroxyanthranilat 4-C-methyl transferase, EC 2.1.1.98 Diphthin synthase, EC 2.1.1.99 3- hydroxy -16-methoxy-2,3-dihydrotabersonine N-methyltransferase, EC 2.1.1.101 macrocin O-methyltransferase, EC 2.1 1.102 demethylmacrocin O-methyltransferase, EC 2.1.1.103 Phosphoethanolamine N-methyltransferase, EC 2.1.1.104 caffeoyl CoA O-methyltransferase, EC 2.1.1.105 N-benzoyl-4-4-Hydroxyanthranilat O-methyl transferase, EG 2.1.1 106 tryptophan 2-C-methyl transferase, EC 2.1.1.107 uroporphyrinogen III methyltransferase C, EC 2.1.1.108 Hydroxymellein 6- O-methyl transferase, EC 2.1.1.109 Demethylsterigmatocystin 6-O-methyl transferase, EC 2.1 .1.110 Sterigmatocystin 8-O- methyl transferase, EC 2.1.1.11 1 anthranilate N-methyltransferase, EC 2.1 ,1.112 glucuronoxylan 4-O-methy! transferase, EC 2.1.1 1 14 Pol yp reny Id i h yd roxy be nzoat methyltransferase, EC 2.1.1.115 (RS)-l-benzyl-l, 2,3,4-tetrahydroisoquinoline N- methyltransferase, EC 2.1.1.116 3'-hydroxy-N-methyl-(S)-Coclaurin 4’-0--methyltransferase, EC 2.1.1.117 (S) 9-0-methyltransferase-Scoulerin , EC 2.1.1.118 Columbamin O- ethyl transferase, EC 2.1.1.119 10- Hydroxydihydrosanguinarin 10-O-methyl transferase, EC. 2 1.1.120 12 Hydroxydihydrochelirubin 12-0- methyltransferase, EC 2.1.1.121 6-0- Methylnorlaudanosolin 5'-0-methyltransferase, EC 2.1.1.122 (S) - tetrahydroprotoberberine N-methyltransferase, EC 2.1.1.123 [cytochrome cj - S- methionine methyl transferase, EC 2.1.1.124 [cytochrome c] arginine N-methyltransferase, EC 2.1.1.128 (RS) - norcoclaurine 6- O-methyi transferase, EC 2.1.1.129 inositol 4-methyl transferase, EC 2.1.1 ,130 Precorrin- 2 C20-methyltransfera$e, EC 2.1.1.131 precorrin-3B C17-methyltransferase, EC 2.1.1.132 precorrin-6Y C5, 15-methyltransferase (decarboxylating), EC 2.1.1 ,133 precorrin-4 Cll- methyltransferase, EC 2.1.1.136 chlorophenol O-methyltransferase , EC 2.1.1.137 arsenite methyltransferase, EC 2.1.1.139 3'Demethylstaurosporin O-methyl transferase, EC 2.1.1.140 (S) -Codaurin-N-methyltransferase , EC 2.1.1.141 jasmonate O-methyl transferase, EC 2.1.1.142 cycloartenol 24- C-methyltransferase, EC 2.1.1.143 24 Methylenesterol C- methyltransferase, EC 2.1.1.144 trans-aconitate 2-methyl transfer ase, EC 2.1.1.145 trans- aconitate 3-methyl transferase, EC 2.1.1.146 (iso) eugenol O-methyltransferase, EC
2.1.1.147 Corydalin synthase, EC 2.1.1.149 myricetin O-methyl transferase, EC 2.1.1.150 isoflavone 7-0- methyltransferase, EC 2.1.1.151 cobalt factor II C20-methyltransferase, EC 2.1.1.152 precorrin-6A synthase (deacetylation), EC 2,1.1.153 vitexin 2 "-O-rhamnoside 7-0- methyi transferase, EC 2.1.1.154 isoliquiritigenin 2 '-O-methyltransferase, EC 2.1.1.155 kaempferol 4'-0-methyltransferase, EC 2.1.1.156 glycine / Sa rcosin N-methyltransferase, EC 2,1.1.157 sarcosine ! dimethylglycine N-methyltransferase, EC 2.1.1.158 7-methylxanthosine synthase, EC 2.1.1.159 theobromine synthase, EC 2.1.1.160 caffeine synthase, EC
2,1.1.161 dimethylglycine N-methyltransferase, EC 2.1.1.162 Glycine i sarcosine / dimethylglycine N-methyltransferase, EC 2.1.1.163 Demethylmenaquinon Methyltransferase, EC 2.1.1.164 Demethylrebeccamycin-D-glucose O-methyl transferase, EC 2.1.1.165 Halide methyl transferase, EC 2.1.1.169 tricetin 3’, 4’, 5 -O-T rimefhyltransferase, EC 2.1.1.175 Tricine synthase, EC 2.1.1.195 cobalt-precorrin-5B (Cl) -methyltransferase, EC 2.1.1.196 Cobalt-precorrin-7 (C15) -methyltransferase, EC 2.1.1.197 malonyl-CoA O- methy!transferase, and EC 2.1.1.201 2-methoxy-6-Polyprenyl- 1, 4-Benzoquinol methylase.
In a preferred embodiment, the alkyltransferase used in step b) transfers the optionally substituted alkyl group by a SN2-type reaction or a radical reaction to the substrate. Alky!transferases, which transfer the optionally substituted alkyl group by a SN2-type reaction are preferred. A SN2-type reaction is nucleophilic substitution, of which the skilled person is familiar with.
The alkyltransferase, which alkylated by an SN2-type reaction is preferably a methyltransferase, preferably a S-adenosyl methionine (SAM) dependent methyltransferase.
The a!kyltransferase, which transfers the optionally substituted alkyl group to the substrate by a SN2-type reaction, is preferably selected from the group consisting of the carminomycin 4- O-methyltransferase DnrK {originates from Streptomyces peucetius,), the demethylrebeccamycin-D-glycose O-methyltransferase RebM (originates from Lechevalieria aerocolonigenes,), the S-adenosyi-L-methionine-dependent O-methyltransferase (SynOMT) (originates from Synechocystis sp. Strain PCC 6803), the O-methyltransferase BcOMT2 (originates from Bacillus cerus), the 2,7-dihydrocy-5-methyl-1-naphthoate 7-0- methyltransferase NcsB1 (originates from Streptomyces carzinostaticus), the phosphonate O-methyltransferase Dhpl (originates from Streptomyces luridus), the demethyldecarbamoylnovobiocin O-methyltransferase NovP (originates from Streptomyces niveus), the methyltransferase CalOI (originates from Micromonospora Echinospora), O- methyltransferase Cal06 (originates from Micromonospora echinospora) the mitomycin 6-0- methyltransferase MmCR (originates from Streptomyces lavendulae), the mycinamicin VI 2”- O-methyltransferase MycE (originates from Micromonospora griseorubida), 10- hyd roxyca m ptot e ci n O-methyltransferase (originates from Camptotheca acuminate), the Norbelladine 49-O-Methyltransferase (originates from Narcissus sp. aff. Pseudonarcissus), the phenazine-1 -carboxylate N-methyltransferase PhzM (originates from Pseudomonas aeruginosa), the dTDP-3-amino-3,4,6-trideoxy-alpha-D-glycopyranose N,N- dimethyltransferase DesVI (originates from Streptomyces fradiae), the NodS N- ethyltransferase (originates from Bradyrhizobium japonicum), the dTDP-3-amino-3,6- d ideoxy-a Ipha-D-g lucopyra nose N,N-dimethyltransferase TylMi (originates from Streptomyces fradiae), the Indolethylamine N-methyltransferase (originates from Homo sapiens), Phenylethanolamine N-methyltransferase (originates from Homo sapiens), beta- alanine N-methyltransferase (originates from Limonium latifolium), dimethylglycine methyltransferase (originates from Galdieria sulphuraria), Ravine N-Methyltransferase (originates from Thalictrum flavum), Psilocybin synthase PsiM (originates from Psilocybe carpophores), Dimethylallyltryptophan N-methyltransferase (originates from Aspergillus fumigatus) , Reticuline N-Methyltransferase (originates from Papaver somniferum), Picrinine N-Methyltransferase (originates from Catharanthus roseus), Norajmaline N- methyltransferase (originates from Rauvolfia serpentine ), (S)-cociaurine-N-methyitransferase (originates from Coptis japonica), the Tetrahydroprotoberberine cis-N-Methyltransferase (originates from Papaver somniferum), the cyclopropane-fatty-acyl-phosphotipid synthase CPFAS (originates from Mycobacterium tuberculosis, Escherichia coli), the methylene-fatty- acyl-phospholipid synthase (originates from Mycobacterium phlei, the uroporphyrinogen-lll C-methyltransferase UMT (originates from Arabidopsis thaiiana), the sterol C- methyltransferase sterol 24-C-methyltransferase (originates from Saccharomyces cerevisiae), and the gamma-tocopherol 5-O-methyltransferase (originates from Arabidopsis thaiiana ).
The alkyltransferase used in step b), which transfer the optionally substituted alkyl group to the substrate by a SN2-type reaction, is preferably N-, C-, or O- specific.
O-specific alkyltransferases are preferably selected from the group consisting of the carminomycin 4-O-methyltransferase DnrK (EC 2.1.1.292; originates from Streptomyces peucetius,), the demethylrebeccamycin-D-glycose O-methyltra nsferase RebM (EC 2.1.1.164; originates from Lechevalieha aerocolonigenes, ), the S-adenosyl-L-methionine-dependent O- methyltransferase (SynOMT) (EC 2.1.1.338 and EC 2.1.1.339; originates from Synechocystis $p. Strain PCC 6803), the O-methyltransferase BcOMT2 (EC 2.1.1.-; originates from Bacillus cerus), the acetylserotonin O-methyltransferase (EC 2.1.1.4; originates from Homo sapiens) the 2,7-dihydrocy-5-methyl-1-naphthoate 7-O-methyltransferase NcsB1 (EC 2.1.1.303; originates from Streptomyces carzinostaticus), the phosphonate O-methyltransferase Dhpl (EC 2.1.1.-; originates from Streptomyces lurid us), the demethyldecarbamoylnovobiocin O- methyltransferase NovP (EC 2.1.1.285; originates from Streptomyces niveus), the methyltransferase CatOI (EC 2.1.1 ,-, originates from Micromonospora Echinospora), O- methyltransferase Cal06 (EC 2.1.1.-, originates from Micromonospora echinospora) the mitomycin 6-O-methyltransferase MmCR (EC 2.1.1.316, originates from Streptomyces iavenduiae), the mycinamicin VI 2”-0-methyltransferase ycE (EC 2.1.1.238, originates from Micromonospora griseorubida), 10-hydroxycamptothecin O-methyltransferase (EC 2.1.1.-; originates from Camptotheca acuminate), the Norbelladine 49-O-Methyltransferase (EC 2.1.1.-; originates from Narcissus sp. aff. Pseudonarcissus) , desmethylxanthohumol o- methyltransferase (EC 2.1.1.338; originates from Carica papaya), xanthohumol 4*-0- methyltransferase (EC 2.1.1.339; originates from Carica papaya), 3-hydroxy-5- methylnaphthoic acid methyltransferase (EC 2.1.1.302; originates from Streptomyces sahachiroi), isoeugenol O-methyltransferase 1 (EC 2.1.1.146; originates from Pimpinella anisum), Chavicol O-methyltransferase (EC 2.1.1.146; originates from Ocimum basilicum), tsoflavone 7-O-m eth yltran sf e ra se (EC 2.11.50; originates from Glycine max), isoflavone 4- O-methyltransferase (EC 2.1.1 48; originates from Pisum sativum), orcino! O- methy!transferase (EC 2.1.1.
Figure imgf000030_0001
originates from Rosa hybrid cultivar), resveratrol O- methyltransferase (EC 2 1.1.240; originates from Vitis vinifera), L-Oleandrosyi 3-0- Methyltransferase (EC 2.1.1.-; originates from Streptomyces antibioticus), Naringenin 7-0- methyltransferase (EC 2 1.1.232; originates from Oryza sativa), indole-3-acetic add carboxyl methyltransferase (EC 2.1.1 278; originates from Arabidopsis thaliana), Gibberellic add methyltransferase (EC 2.1.1 278; originates from Arabidopsis thaliana), salicylic add carboxyl methyltransferase (EC 2 1 ,1.274; originates from Petunia hybrida), Jasmonic acid carboxyl methyltransferase (EC 2.1.1.141 ; originates from Arabidopsis thaliana), Akalonic acid O-methyltransferase (EC 2.1.1.-; originates from Streptomyces peucetius), fatty-acid O- methyltransferase (EC 2.1.1.15; originates from Mycobacterium marinum).
N-specific alkyltransferases are preferably selected form the group consisting of the phenazine-1 -carboxylate N-methyltransferase PhzM (EC 2 1.1 327; originates from Pseudomonas aeruginosa), the dTDP-3-amino-3,4,6-trideoxy-alpha-D-glycopyranose N,N- dimethyltransferase DesVI (EC 2.1.1 ,234; originates from Streptomyces fradiae), the NodS N-methyltransferase (EC 2.1.1.-; originates from Bradyrhizobium japonicum), originates from Bradyrhizobium japonicum, N-specific), the dTDP-3-amino-3,6-dideoxy-alpha-D- glucopyranose N,N-dimethyltransferase TylM1 (EC 2.1.1.235 and EC 2.1.1.236; originates from Streptomyces fradiae), the Indolethylamine N-methyltransferase (EC 2.1.1.96 and 2.1.1.49; originates from Homo sapiens), Phenylethanolamine N-methyltransferase (EC 2.1.1.?; originates from Homo sapiens), beta-alanine N-methyltransferase (EC 2.1.1.-; originates from Limonium latifolium), dimethylglydne methyltransferase (EC 2.1.1.157; originates from Galdieria sulphuraria), glycine/sarcosine N-methyltransferase (EC 2.1.1.156; originates from Pseudomonas fiuorescens) Pa vine N-Methyltransferase (EC 2.1.1.300; originates from Thalictrum flavum), Anthranilate N-methyltransferase (EC 2.1.1.11 1 ; originates from Ruta graveolens) Psilocybin synthase PsiM (EC 2.1.1.345; originates from Psiiocybe carpophores), Dimethylallyltryptophan N-methyltransferase (EC 2.1.1.261 ; originates from Aspergillus fumigatus), Reticuline N-Methyltransferase (EC 2.1.1.337; originates from Papaver somniferum), 3-aminomethylindole/N-methyl-3-aminomethylindole N-methyltransferase (EC 2.1.1.-; originates from Hordeum vulgare L ), Picrinine N- Methyltransferase (EC 2.1.1.-; originates from Gatharanthus roseus), Norajmaline N- methyltransferase (EC 2.1.1.-; originates from Rauvolfia serpentine), (S)-coclaurine-N- methyltransferase (EC 2.1.1.140; originates from Coptis japonica), the
Tetrahydroprotoberberine cis-N-Methy!transferase (EC 2.1.1122; originates from Papaver somniferum), N-demethy!lincomycin N-methyltransferase (EC 2,1.1328; originates from Streptomyces lincolnensis), Trigonelline synthase (EC 2.1.1. 7; originates from Trigonella foenum-graceum) , and Caffeine synthase (EC 2.1.1. 160; originates from Coffea arabica).
C-specific alkyltransferases are preferably selected form the group consisting of the cyclopropane-fatty-acyl-phospholipid synthase CPFAS (EC 2.1.179; originates from Mycobacterium tuberculosis, Escherichia colt), the methylene-fatty-acyl-phospholipid synthase (EC 2,1.116, originates from Mycobacterium phlei, the uroporphyrinogen-ill C- methyltransferase UMT (EC 2.11.107; originates from Arabidopsis thaliana), the sterol C- methyltransferase sterol 24-C-methy!transferase (EC 2.1.1.142; originates from Saccharomyces cerevisiae), the gamma-tocopherol 5-O-methyltransferase (EC 2.1.1.95; originates from Arabidopsis thaliana), L-tyrosine C(3)-methyltransferase (EC 2.1.1304; originates from Streptomyces lavendulae), triterpene methyltransferase 1 (EC 2.1.1 262; originates from Botryococcus braunii), triterpene methyltransferase 2 (EC 2.1.1. 262; originates from Botryococcus braunii), triterpene methyltransferase 3 (EC 2.1.1 263; originates from Botryococcus braunii), Geranyl Diphosphate C-Methyltransferase (EC 2.11 255; originates from Streptomyces coelicoior), D-mycarose 3-C-methyltransferase (EC 2.1.1. -; originates from Streptomyces argillaceus), 5-guanidino-2-oxo-pentanoic acid methyltransferase EC 2.1.1 originates from Pseudomonas syringae), Indolepyruvate C- methyltransferase (EC 2.1.1.47; originates from Streptomyces griseus).
More preferably, the alkyltransferase used in step b) is selected from the group consisting of the histidine-specific methyltransferase EgtD (SEQ ID NO: 2), EC 2.1144), the inositol 4- methyltransferase (IMT, SEQ ID NO: 3) (EC 2.1.1.129), the putrescine N-methyl transferase (PMT, SEQ ID NO: 4) (EC 2.11.53), the methyltransferase SgvM (SEQ ID NO: 5, EC 2.1.1), the 8-demethylnovobiocic acid CS-methyttransferase NovO (SEQ ID NO: 6, EC 2.1.1.284) and the 5-thiohistidine N-methyltransferase OvoC (SEQ ID NO: 7, EC 2.1.1.-).
The histidine-specific methyltransferase EgtD (EC 2.1.1.44) originates from Mycobacterium smegmatis, is N-specific and uses histidine as substrate. The histidine methyltransferase EgtD is a SAM-dependent methyltransferase, which can catalyzes trimethylation of the alpha-amino group of histidine. The resulting N-alpha-trimethyl histidine (TMH) is a precursor for ergothioneine biosynthesis in bacteria and fungi.
The inositol 4-methyltransferase originates from Mesembryanthemum crystallinum, is O- specific and uses inositol as substrate. Inositol 4-methyltransferase (IMT) (EC 2.11.129) converts inositol to ononitol. O-Methyl inositols are plant osmoprotectants, which have antidiabetic, anti-cancer or anti-inflammatory effects in humans. Inositol methyltransferases reflect the synthetic process of enzyme-mediated methylation in that they afford regiospecific alkylation of one out of six nearly equivalent hydroxyl groups. Chemical approaches for the same transformation include laborious multistep syntheses.
The putrescine N-methyl transferase (PMT) (EC 2.1.1.53) orginates from Anisodus tanguticus, is N-specific and uses diamine putrescine as substrate. The utrescine N-methyl transferase (PMT, EC2.1.1.53) can catalyze the methylation of the diamine putrescine to form the first intermediate in the biosynthesis of alkaloids, including nicotine and tropanes. Transformation of primary amines to secondary amines by chemical synthesis is often hampered by the higher nucleophilicity of the secondary amine which results in overalkylation. Hence, secondary methyl amines are usually synthesized through indirect routes. In contrast, PMT avoids over-methyiation by way of its carefully tailored active site.
C-Methylating enzymes such as the methyltransferase SgvM and the 8-demethylnovobiocic acid C8-methyltransferase NovO are notable for their abilities of asymmetric alkylation of the beta-carbon of alpha-keto acids. This activity is of particular interest, since direct asymmetric alkylation alpha to ketones is still a challenging objective for synthetic methods. The methyltransferase SgvM (EC 2.1.1.-) originates from Streptomyces griseoviridis, is C-specific and uses alpha-ketonorvaline and other alpha-keto acids as substrate. The methyltransferase SgvM can methylate the C3 position of alpha-ketonorvaline, among other substrates. The 8-demethylnovobiocic acid C8-methyltransferase NovO (EC 2.1.1.284) originates from Streptomyces niveus, is C-specific and uses 8-demethylnovobiocic as substrate. NovO can methylate the sp2-hybridized carbon on the aromatic core of coumarins.
The 5-thiohistidine N-methyltransferase OvoC (EC 2.1.1.-) originates from Erwinia tasmaniensis, is N-specific and uses 5-thiohistidine as substrate. OvoC can transfer a methyl group from S-adenosyl methionine (SAM) to produced ovothiol A.
In a further embodiment, the alky!transferase used in step b) transfers the optionally substituted alkyl group by a radical reaction. In a radical reaction, the alkyl group may be transferred in the form of a free radical, thus comprising an unpaired electron. Such alkyltransferases are also referred to as radical alkyltransferases. These enzymes transfer methyl radicals to the substrates. The radical aikyltransferase is preferably a methyltransferase, more preferably an S- adenosyl methionine (SAM) dependent methyltransferase.
The aikyltransferase, which transfers the optionally substituted alkyl group to the substrate by a radical reaction, is preferably selected from the group consisting of Tryptophan 2-C- methyltransferase TsrM, P-methyltransferase, Valine methyltransferases PoyC, CioN6, Pyrrole-2-carboxyi methyltransferase, Fosfomycin Biosynthesis Enzyme Fom3, GenK, Gentamicin biosynthetic methyltransferase.
The tryptophan 2-C-methyltransferase TsrM originates from Streptomyces laurentii, and is C- specific. The P-methyltransferase originates from Streptomyces viridochromogenes, is P- specific and uses N-Acetyldemethylphosphinothricin tripeptide as substrate. The valine methyltransferases PoyC originates from the bacterium symbiont of Theonella swinhoei pTSMAd , is C-specific and uses the peptide PoyA (SEQ ID NO: 8) as substrate.
The Fosfomycin Biosynthesis Enzyme Fom3 originates from Streptomyces wedmorensis, is C-specific and uses (5'-cytidylyl)-2-hydroxyethylphosphonate as substrate. The GenK, Gentamicin biosynthetic methyltransferase originates from Micromonospora echinospora, is C-specific and uses gentamicin X2 as substrate.
Several radical methyltransferases consume two equivalents of S-adenosyl methionine (SAM) per methyl transfer. A first equivalent is converted to S-adenosyl homocysteine (SAH), a second equivalent is converted to methionine and 5-deoxy adenosine (5dAdo). Therefore, SAM needs to be regenerated by two different systems: i) SAH could be recycled by as described above, for example by a halide methyl transferase and methyl halide; ii) Methionine could also be recycled by adenosyl-chloride synthase (chlorinase) using the (synthetic) co-substrate 5-chloro-5-deoxy adenosine (5-CI-dAdo), Overall this method uses three enzymes: a halide methyl transferase as aikyltransferase in step a), a radical methlytransferase in step b) and a chlorinase. The reaction consumes methyl halide, 5-CI- dAdo and substrate and produces methylated product, chloride and 5-deoxy adenosine (5dAdo).
Thus, in one embodiment, wherein the aikyltransferase used in step b), which transfers the optionally substituted alkyl group by a radical reaction, is a SAM-dependent aikyltransferase, preferably a SAM-dependent methyltransferase, and the sulfur-containing carrier compound is S-adenosyl homocysteine (SAH), step b) additional yields methionine. In this embodiment, at least a part of the methionine yielded in step b) is regenerated by an adenosyl-chloride synthase (chlorinase), which synthesizes S-adenosyl-L-methionine (SAM) from methionine and a co-substrate. The co-substrate for regenerating methionine is preferably 5-chloro-5- deoxy adenosine.
Converting enzymes
Converting enzyme according to the present invention convert a substrate into a product, or a presubstrate into a substrate.
The converting enzyme used in step c) is an enzyme that converts the beta-methylated substrate yielded in step b) to a beta-methylated product. The converting enzyme used in step c) is preferably a transaminase or an isomerase.
The converting enzyme used in step d) is an enzyme that converts a presubstrate to the substrate that is alkylated in step b). The converting enzyme used in step d) is preferably selected from the group consisting of a transaminase, an alcohol dehydrogenase and an alpha-keto acid decarboxylase.
A transaminase is an enzyme that catalyzes the amino transfer, for example between a a- keto acid and an amino acid, or between a primary amine and an aldehyde (see Figures 37 and 46 C). An amino acid contains an amine (NH2) group. A keto acid contains a keto (=0) group. The transamination, the amine (NH2) group on one molecule is exchanged with the keto (=0) group on the other molecule. The amino acid is converted to a keto acid, and the keto acid is converted an amino acid. The transaminase can use amino acids or proteins as substrate. Preferably, a transaminase uses the cofactor pyridoxal-phosphate (PLP). PLP is converted into pyridoxamine, when an amino acid is converted into a keto acid. Enzyme- bound pyridoxamine in turn reacts with pyruvate, oxaloacetate, or alpha-ketoglutarate, giving alanine, aspartic acid, or glutamic acid, respectively. The reaction catalyzed by a transaminase is also called transamination.
An isomerase is an enzyme that catalyzes changes within one molecule. An isomerease can convert one isomer to another. An isomer has the same molecular formula but a different physical structure. An isomer can be a structural isomers or stereoisomer. Structural isomers have a different ordering of bonds and/or different bond connectivity from one another. Stereoisomers have the same ordering of individual bonds and the same connectivity but the three-dimensional arrangement of bonded atoms differ. Preferred isomerases are selected from the group consisting of racemases and epimerases. Cis-trans isomerases are examples of enzymes catalyzing the interconversion of stereoisomers. Intramolecular lyases, GXidoreductases and transferases examples of enzymes that catalyze the interconversion of structural isomers. The reaction catalyzed by an isomerase is also called isomerization. See also Figure 37.
An alcohol dehydrogenase (ADH; EC 1.11.1 ) is an enzyme that catalyzes the conversion between alcohols and aldehydes or between alcohols and ketones. The reaction catalyzed by ADH is preferably a redox reaction. Alcohol dehydrogenase may use nicotinamide adeine dinucleotide (NAD+) as cofactor, which is reduced to NADH during the reaction. See also Figure 46 A and B.
An alpha-keto acid decarboxylase is an enzyme that catalyzes the conversion between ketones and alpha-keto acids. See also Figure 46 B and C.
Alkyl donor and optionally substituted alkyl group
In the method of the present invention, the S-a!kyltransferase transfers an optionally substituted alkyl group from an alkyl donor to the sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound.
As will be understood by a person skilled in the art, the alkyl donor is a compound which donates the optionally substituted alkyl group. Thus, the alkyl donor is a compound comprising an optionally substituted alkyl group, and is preferably a compound comprising an optionally substituted alkyl group and a leaving group. Typically, the alkyl donor is a compound which donates one alkyl group, i.e. a compound which comprises one optionally substituted alkyl group which is amenable to being transferred from the alkyl donor to a carrier compound by the alkyltransferase in the alkylation method or the carrier production method in accordance with the invention.
In accordance with the present invention, the optionally substituted alkyl group of the alkyl donor is transferred in step a) to the sulfur- or selenium-containing carrier compound and is further transferred in step b) to the substrate, thereby alkylating the substrate. In other words, the optionally substituted alkyl group which is transferred in step a) and the optionally substituted alkyl group which is transferred in step b) have the same structure.
The alkyl part of the optionally substituted alkyl group as referred to herein has preferably 1 to 5 carbon atoms, more preferably 1 to 3 carbon atoms and still more preferably 1 carbon atom. The optionally substituted alkyl group may be substituted by one or more, such as one, two or three, substituents. Examples of the substituents include halogen atoms, such as a chlorine atom or a fluorine atom, an alkenyl group, preferably an alkenyl group with 2 to 5 crbon atoms, such as a vinyl group, and an alkinyl group, preferably an alkenyl group with 2 to 5 carbon atoms, such as an ethinyl group.
Examples of the optionally substituted alkyl group include a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyl group, a difluoromethy! group and a trifluoromethyl group. Further examples of the optionally substituted alkyl group include a 2-fluoroethyl group, 2,2-difluoroethyl group and 2,2,2-trifIuoroethyl group.
The optionally substituted alkyl group may be an alkyl group which is not substituted. As above, the alkyl group has preferably 1 to 5 carbon atoms, more preferably 1 to 3 carbon atoms and still more preferably 1 carbon atom.
Preferably, the alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group. More preferably, the atkyl group is a methyl group.
Preferably, the optionally substituted alkyl group is a methyl group or a monofluoromethyl group. More preferably, the unsubstituted alkyl group is a methyl group. The substituted alky! group is more preferably a monofluoromethyl group.
It is noted that the optionally substituted alkyl group may be isotopically labeled with an isotope selected from the group consisting of 1eF, 2H (deuterium), 11C, 13C, and 14C. Thus, any fluorine atom, any hydrogen atom and any carbon atom of the optionally substituted alkyl group may be replaced by its isotope. Isotopically labeled alkyl groups may be used to isotopically label a substrate, preferably in vitro or in vivo
A leaving group is a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage, A leaving group may be an anion or a neutral molecule. Exemplary leaving groups are halide ions such as iodide, bromide and chloride; sulfate; cyclic thioethers such as tetrahydrothiophen, thietane and ethylene sulphide; linear thioethers such as dialkyl sulphide (alkyl = C1-C4 alkyl) and methionine; and selenoethers such as selenomethionine. The leaving group is preferably selected from the group consisting of iodide, bromide, chloride, and methionine; more preferably selected from the group consisting of iodide and chloride; still more preferably iodide.
In the method of the present invention, the alkyl donor is preferably a halide alkyl donor, a sulfur-based alkyl donor, an oxygen-based alkyl donor or a selenium-based alkyl donor. The halide alkyl donor, which may also be referred to as an alkyl halide, is preferably a methyl halide (e.g. methyl iodide or monofluoromethyl iodide). The sulfur-based alkyl donor is preferably selected from an S-alkylated thioether (e.g. S-methylmethionine or S- methyltefrahydrothiophen) or an S-alkylated sulfoxide (e.g. a trimethylsulfoxonium ion, such as trimethylsulfoxonium chloride, bromide or iodide). The oxygen-based alkyl donor is preferably selected from an O-alkylated carbonate, carbamate or thiocarbonate. The selenium-based alkyl donor is preferably selected from an Se-aikylated selenoether (e.g. Se~ methylselenomethionine or Se-methyltetrahydroselenophene), an Se-alkylated selenoxide (e.g. a trimethylselenoxonium ion, such as trimethylselenoxonium chloride, bromide or iodide) or a trialkylselenonium (e.g. a trimethylselenonium ion, such as trimethylselenonium chloride, bromide or iodide).
Suitable examples of the alkyl donor include methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, propargyl bromide, propargyl chloride, S-methyl methionine, Se-methyl selenomethionine, methyl sulfate, S-methyltetrahydrothiophene, S-methyl thietane, S-methyl ethylene sulphide, S-methyl di(monofluoromethyl) sulphide, S-methyl dimethyl sulphide, S~ methyl 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid and S-methyl 3- (methylthio)propionic acid. Further suitable examples of the alkyl donor include monofluoromethyl iodide, 1 -monofluoro-2-iodoethane, 1 , 1 -d if luoro-2-iodoethane , 1 ,1 ,1- trifluoro-2-iodoethane, trimethylselenonium iodide, S-methyl-fluoromethionine (fluoromethionine = 2-amino-4-(f!uoro-methylthio)butanoic acid) and Se-methyi- selenofluoromethionine (fluoroselenomethionine = 2-amino-4-(fluoro-methylseleno)butanoic acid).
Preferred examples of the alkyl donor are methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, S-methyl methionine and Se-methyl selenomethionine.
More preferred examples of the alkyl donor are methyl iodide, methyl bromide, methyl chloride and S-methyl methionine, still more preferably methyl iodide, methyl bromide and methyl chloride, most preferably methyl iodide. Further most preferred examples of the alkyl donor are monofluoromethyi iodide and Se-methyl selenomethionine.
The alkyl donor being an oxygen-based alkyl donor is preferably represented by the following formula (II):
Figure imgf000038_0001
wherein R10 is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyi group, a difluoromethyl group and a trifluoromethyl group, preferably a methyl group; X is selected from the group consisting of NH, O and S; and R10 is selected from an alkyl group or an aryl group, preferably a methyl group or ethyl group.
The alkyl donor of formula (II) is preferably dimethylcarbonate. Advantages of dimethylcarbonate as alkyl donor are that the alkytdonor produces only volatile side products, and dimethylcarbonate is less toxic and less expensive than methyliodide.
Without wishing to be bound by theory, it is assumed that the alkyl donor of formula (II) such as dimethylcarbonate reacts with the sulfur- or selenium-containing carrier compound such as SAH to produce an alkylated sulfur- or selenium-containing carrier compound such as SAM, R11XH such as methanol and carbon dioxide.
Most preferably, the alkyl donor according to the present invention is a methyl donor. In this most preferred case, the methyl group of the methyl donor is transferred in step a) to the sulfur- or selenium-containing carrier compound and is further transferred in step b) to the substrate, thereby methylating the substrate.
Examples of the most preferred methyl donor are methyl iodide, methyl bromide, methyl chloride and S-methyl methionine, still more preferably methyl iodide, methyl bromide and methyl chloride, most preferably methyl iodide. Further examples of the most preferred methyl donor is monofluoromethyi iodide and Se-methyl selenomethionine.
It will be appreciated by the skilled reader that the alkyl donor used in the alkylation method and the carrier production method according to present invention differs from the carrier compound used in these methods. In particular, the alkyl donor is not S-adenosyl methionine (SAM)
In the alkylation method and the carrier production method in accordance with the present invention, the alkyl donor is preferably used in an amount which is at least stoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate. Preferably, the alkyl donor is used in an amount corresponding to 1.0 moles, more preferably to 1.5 moles of the alkyl donor, for each mole of optionally substituted alkyl group to be transferred to the substrate.
Substrate
In accordance with the present invention, the S-aikyltransferase transfers the optionally substituted alkyl group from the alkyl donor to the sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound; and further a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound.
As will be understood by the skied reader, the substrate (also referred to as acceptor) is an organic compound with a suitable atom suitable for alkylation, such as a sulfur atom, nitrogen atom, a carbon atom, oxygen atom or phosphorus atom. The substrate according to the present invention preferably comprises at least one nucleophilic atom selected from the group consisting of a nucleophilic nitrogen atom, a nucleophilic carbon atom, a nucleophilic oxygen atom, a nucleophilic sulfur atom and a nucleophilic phosphorus atom. More preferably, it comprises at least one nucleophilic atom selected from the group consisting of a nucleophilic nitrogen atom, a nucleophilic carbon atom, and a nucleophilic oxygen atom.
The substrate preferably comprises a C-H acidic carbon-hydrogen bond or a group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group and phosphine group, more preferably a primary amino group, a secondary amino groups, a hydroxy group, an amide group, and a carboxy group. The substrate may preferably also comprise a C-H acidic carbon-hydrogen bond and/or a group selected from the group consisting of a primary amino group, a secondary amino group, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a phosphonate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group, phosphine group, a carbonyl group and combinations thereof, more preferably a primary amino group, a secondary amino groups, a hydroxy group, an amide group, a carboxy group, a carbonyl group and combinations thereof,
As examples for a nucleophilic carbon atom comprising a C-H acidic carbon-hydrogen bond, a carbon atom in alpha position to a carbonyl group or to a nitro group, or an aromatic carbon atom in ortho position to a hydroxy group may be mentioned.
Examples of the substrate prior to alkylation and/or of the alkylated substrate include a small molecule with a molecular mass of less than 1 kDa (1000 g/mol), primary metabolites, secondary metabolites, peptides such as ribosomal peptides, proteins, polysaccharides and nucleic acids.
The small molecule may have a molecular mass of less than 0,9 kDa (900 g/mol), preferably less than 0.8 kDa (800 g/mol). Small molecules as defined herein may also be primary metabolites and/or secondary metabolites.
Metabolite refers to intermediate or end products of the metabolism of a cell. Primary metabolites may be directly involved in cell growth, development and reproduction. Secondary metabolites may be indirectly involved in these processes. The primary metabolite is preferably selected from the group consisting of amino acids (such as histidine), nucleosides, nucleotides, lipids, carbohydrates. The secondary metabolite is preferably selected from the group consisting of non-ribosomal peptides, polyketides, terpenes, alkaloids, phenylpropanoids, purtn- and pyrimidine-based metabolites, and antibiotics. Specific examples of the substrates prior to alkylation include histidine, inositol, putrescine, a-ketonorvaline, and coumarine. Specific examples of substrates after alkylation include L- hercynine, ononitol, A/,W-dimethyl-1 ,4-butanediamine, R-3-methyl-2-oxovalerate, dihydroxy- 1-methylnaphthalene and ovothiol.
It will be appreciated that the substrate which is alkylated in step b) of the alkylating method in accordance with the invention differs from the carrier compound. In particular, the substrate which is alkylated in step b) is not S-adenosyl methionine.
The alkylated substrate, also referred to as the product or alkylated acceptor, is the substrate which comprises at least one optionally substituted alkyl group. As should be understood by a person skilled in the art, the at least one optionally substituted alkyl group of the alkylated substrate is covalently bound to a nitrogen atom, a carbon atom, an oxygen atom, a sulfur atom and/or a phosphorus atom of the substrate.
The alkylated substrate may be isolated and optionally purified, The alkylated substrate may be isolated from the cell-free system or buffer if it was produced in vitro. Alternatively, the alkylated substrate may be isolated from a celt if it was produced in vivo. The cells may be lysed by any technique known to the skilled person, and purified from the supernatant after removing cell debris by centrifugation.
In a further embodiment, the alkylated substrate is further converted to an alkylated product. In that case, the method is preferably used for b-methylation of L- or D-a-amino acids, L- or D-a-hydroxy acids, amines or alcohols, preferably for b-methylation of L- or D-a-amino acids. Here, the alkylated substrate yielded in step b) is a b-methylated substrate (for example b- methyl a-keto acid), which is further converted to a b-methylated product (for example b- methyl a-amino acid, or b-methyl a-hydroxy acid, or 2-methyl amine, or 2-methyl alcohol) by at least one additional step;
a conversion step c) wherein a converting enzyme converts the b-methylated substrate to a b-methylated product. The b- ethylated product is preferably selected from the group consisting of a b-methyl-L-a-amino acid, a b-methyl-D-a-amino acid, a b-methyl-L-a-hydroxy acid, a b-methyl-D-a-hydroxy acid, a 2-methyl-amine and a 2-methyl-alcohol. If the b- methylated substrate is a b-methyl-a-keto acid and the b-m ethylated product is selected from the group consisting of a b-methyi-L-a-amino acid and a b-methyl-D-a-amino acid, conversion step c) is a transamination wherein the converting enzyme is a transaminase that converts the b-methyl-a-keto acid to a b-m ethyl-ami no acid as the b-methylated product. The method may comprise a further conversion step d ) after step b) and before step c), wherein step d ) is an isomarization step wherein an isomerase converts one isomere of the b- methylated et-keto acid to a different isomere. See also Figure 37 and 47.
Optionally, the method described herein may comprise at least one additional step:
a conversion step d) wherein at least one converting enzyme converts a presubstrate to the substrate. See also Figure 37 and 47. Here, the substrate that is alkylated may be obtained from a presubstrate. The presubstrate is a precursor of the substrate. The presubstrate may be converted to the substrate by at least one converting enzyme in at least one additional conversion step d), which is preferably carried out before step b). Examples of the presubstrate include amino acids, alpha-hydroxy acids, alpha-keto acids, amines, alcohols or ketones, preferably amino acids, alpha-hydroxy acids or ketones. Alpha-hydroxy acids can additionally be obtained from alpha-keto acids. Ketones can additionally be obtained from amines or alcohols. Examples of the converting enzyme used in step d) are transaminase, alcohol dehydrogenase, or alpha-keto acid decarboxylase. For example, an amino acid as presubstrate can be converted to an alpha-keto acid as substrate by a transaminase as converting enzyme in step d). An alpha-hydroxy acid as presubstrate can, for example, be converted to an alpha-keto add as substrate by alcohol dehydrogenase as converting enzyme in step d). Optionally, the alpha-hydroxy add presubstrate may additionally be obtained from an alpha-keto acid by an alcohol dehydrogenase as additional converting enzyme. A ketone as presubstrate can, for example be converted to an alpha-hydroxy acid as a substrate by alpha-keto-acid decarboxylase as converting enzyme in step d). Optionally, the ketone as presubstrate may additionally be obtained from an alcohol by an alcohol dehydrogenase as additional converting enzyme or the ketone may additionally be obtained from an amine by a transaminase as additional converting enzyme. See also Figures 37 and 46.
In line with the above, the method for alkylating a substrate in accordance with a particularly preferred embodiment of the invention comprises the following steps:
a) an alkylation step wherein a S-adenosyl methionine (SAM) dependent S-alkyltransferase transfers an alkyl group from an alkyl donor to a sulfur-containing carrier compound of formula (la), yielding an alkylated sulfur-containing carrier compound of formula (la);
b) an alkylation step wherein a SAM dependent N-, C-, or O-alkyltransferase transfers the alkyl group from the alkylated sulfur-containing carrier compound of formula (la) to the substrate, yielding an alkylated substrate and a dealkyiated sulfur-containing carrier compound of formula (la);
wherein at least a part of the dealkyiated sulfur-containing carrier compound of formula (la) yielded in step b) is recycled to step a) to regenerate the alkylated sulfur-containing carrier compound of formula (la);
and wherein the sulfur-containing carrier compound of formula (la) is preferably S-adenosyl homocysteine (SAH). The preferred optionally substituted alkyl group is also in this context a methyl group.
In a still further preferred embodiment, the method for alkylating a substrate is a method for methylating a substrate comprising the following steps:
a) a methy!ation step wherein a halide methyl transferase (HMT) transfers a methyl group from methyl halide used as methyl donor to S-adenosyl homocysteine (SAH) used as carrier compound, yielding S-adenosyl methionine (SAM);
b) a methylation step wherein a SAM dependent N-, C-, or O-methyltransferase transfers the methyl group from SAM to the substrate, yielding a methylated substrate and SAH;
wherein at least a part of SAH yielded in step b) is recycled to step a) to regenerate SAM; and wherein methyl halide is preferably methyl iodide or methyl chloride, more preferably methyl iodide.
Kit for alkylating a substrate
The present invention further relates to a kit for alkylating a substrate comprising a S- alkyltransferase as defined herein above, a N-, C-, O-, S-, or P-alky!transferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above. The S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase comprised in the kit. The N-, C-, 0-, S-, or P-alkyltransferase is an alkyltransferase as used in step b) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step b) also relate to the N-, C-, 0-, S-, or P-alkyltransferase comprised in the kit.
The kit for alkylating a substrate may further comprise a substrate as defined herein above. Here, the substrate is a substrate prior to alkylation.The kit for alkylating the substrate may further comprise a presubstrate and at least one converting enzyme, which converts the presubstrate to the substrate. The presubstrate and the converting enzyme are as defined herein above.
The kit for alkylating a substrate is preferably substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3,2.2.9). A kit substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) in the context of the present invention means that the kit comprises less than 5%, preferably less than 2%, more preferably less than 1 %, even more preferably less than 0.1% S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2 9). Furthermore, in a kit, which is substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9), the activity of the S-adenosyl homocysteine (SAH) nucleosidase may be less than 0.05% mol units per mg alkyltransferase, preferably 0.025% mol units per mg alkyltransferase, more preferably less than 0.013% mol units per mg alkyltransferase; and/or the activity of the S-adenosyl homocysteine (SAH) nucleosidase may less than 1 pmol/min per mg alkyltransferase, preferably 0.5 pmo!/min per mg alkyltransferase, more preferably less than 0.25 pmol/min per mg alkyltransferase.
In a further embodiment, the kit for alkylating a substrate further comprises an adenosyl- chloride synthase and a co-substrate, which is preferably 5-chloro-5-deoxy adenosine.
The alkylated substrate may be isolated and optionally purified as described above.
In particularly preferred embodiment, the kit for alkylating a substrate comprises a S- methyltransferase as defined herein above for use in step a) of the alkylation method in accordance with the invention, a N-, C-, O-, S-, or P-methylitransferase as defined herein above for use in step b) of the alkylation method in accordance with the invention, a methyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above. In this embodiment, the kit is for methylating a substrate. The kit for methylating a substrate may comprise a halide methyl transferase as S-methyltransferase, the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase IMT, the putrescine N-methyltransferase PMT, or the 8-demethylnovobiocic acid C8- methyltransferase NovO as N-, C-, O-, S-, or P-methyltransferase; methyl iodide as a methyl donor and S-adenosyl homocysteine (SAH) as sulfur- or selenium-containing carrier. The kit for methylating a substrate may also comprise the methionine S-methyltransferase ( SMT) as S-m eth ltra n sfe ra s e , the 5-thiohistidine N-methyltransferase OvoC as N-, C-, 0-, S-, or P- methyltransferase, S-methyl methionine (SMM) or Se-methyl-selenomethionine (SeMSeM) or S-methyl-fluoromethionine (fluoromethionine = 2-amino-4-(fluoro-methy!thio)butanoic acid) or Se-methyl-selenoffuoromethionine (fluoroselenomethionine - 2-amino-4-(fluoro- methy!seleno)butanoic acid) as a methyl donor and S-adenosyl homocysteine (SAH) as sulfur- or selenium-containing carrier.
Use for the production of an alkylated substrate
The present invention also relates to the use of a S-alkyltransferase as defined herein above, a N-, C-, 0-, S-, or P-alkyltransferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above for the production of an alkylated substrate. The S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase used for the production of an alkylated substrate. The N-, C-, 0-, S-, or P-alkyltransferase is an alkyltransferase as used in step b) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step b) also relate to the N-, C-, 0-, S-, or P-alkyltransferase used for the production of an alkylated substrate.
The present invention further relates to the use of a kit as defined herein above for the production of an alkylated substrate.
Any substrate may be alkylated. The substrates prior to alkylation as described herein above are preferred. The alkylated substrate is preferably as described herein above. In a preferred embodiment, the alkylated substrate is isotopically labeled. In this embodiment, an isotopically labeled alkyl donor as defined herein above is preferred.
The alkylated substrate may be produced in vitro or in vivo. Any limitations described above for the method for alkylating a substrate also apply to the use for the production of an alkylated substrate.
The alkylated substrate is preferably produced in the absence of S-adenosyl homocysteine
(SAH) nucleosidase (EC 3.2.2.9).
After production of the alkylated substrate, the alkylated substrate may be isolated and optionally purified as described herein.
Method for producing an alkylated carrier compound
The present invention also relates to a method for producing an alkylated sulfur- or selenium- containing carrier compound, comprising an alkylation step, wherein a S-alkyltransferase as defined herein above transfers an optionally substituted alkyl group as defined herein above from an alkyl donor as defined herein above to a sulfur- or selenium-containing carrier compound as defined hereinabove, yielding an alkylated sulfur- or selenium-containing carrier compound. The S-alkyftransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step a) also relate to the S-a!kyltransferase used in the method for producing an alkylated carrier compound. The obtained alkylated sulfur- or selenium- containing carrier compound is preferably as defined herein above.
In a preferred embodiment a methylated sulfur- or selenium-containing carrier compound is produced. In this preferred embodiment, the optionally substituted alkyl group is a methyl group. It is preferred that the alkylated sulfur- or selenium-containing carrier compound produced by the method is a methylated sulfur-containing carrier compound, more preferably S-adenosyl-methionine (SAM). The sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
The S-alkyltransferase which is preferably used in the method for producing S-adenosyl- methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum. If a halide methyl transferase is used, the alkyl donor is preferably methyl iodide, methyl bromide or methyl chloride, preferably methyl iodide or methyl chloride, more preferably methyl iodide.
It is also preferred to use a methionine S-methyltransferase (MSMT), preferably a methionine S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp. JA431, as S~ alkyltransferase in the method for producing S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound. If a methionine S-methyltransferase (MSMT) is used, the alkyl donor is preferably S-methyl methionine or Se-methyl selenomethionine.
Most preferably, the method is for producing S-adenosyl-methionine (SAM) using S-adenosy! homocysteine (SAH), a halide methyl transferase as S-alkyltransferase and methyl iodide as alkyl donor. Also preferred is a method for producing S-adenosyl-methionine (SAM) using S- adenosyl homocysteine (SAH), a methionine S-methyltransferase as S-alkyltransferase and S-methyl methionine as alkyl donor.
The method for producing an alkylated sulfur- or selenium-containing carrier compound, is preferably carried out in the absence of S-adenosyt homocysteine (SAH) nucleosidase (EC 3 2.2.9). The absence of the S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2, 2.9) is preferred for the reasons explained herein above.
The alkylated carrier compound may be produced in vitro or in vivo.
If the method is carried out in vitro, it is preferably carried out in a cell free-system or a buffer. A cell-free system may be an extract from a cell, which expresses the S-alky!transferase. The cell extract can be obtained by lysis of the cell. If the method is carried out in a buffer, the S-a!kyltransferase is obtained from a cell, for example by purification, and added to the buffer. A typical buffer is a sodium phosphate buffer, such as a 100 mM sodium phosphate buffer with a pH of 8.0.
If the carrier production method in accordance with the invention is carried out as an in vitro method, the alkyl donor, the sulfur- or selenium-containing carrier compound and the S- alkyltransferase are typically combined in a single reaction vessel, before allowing the reaction to proceed.
If the method is carried out in vivo, it is preferably carried out in a host cell. The host cell is preferably a non-human or non-animal cell, more preferably a bacterial cell, plant cell or fungal cell. Particularly preferred bacterial cells are E.coli and C. Giutamicum. The skilled person is familiar with suitable host cell and expression systems. For example, a gene encoding an alkyltransferase of the present invention may be cloned into a vector under control of a promoter such as an inducible promoter. After induction of the promoter, the gene is expressed and the method can be carried out.
It is preferred that the method is carried out in the absence of S-adenosyl adenosine nucleosidase (EC 3.2.2.9), when S-adenosyl homocysteine is used as a carrier compound. If the method is carried out in vivo, the host cell is preferably S-adenosyl adenosine nucleosidase (EC 3.2.2.9) deficient.
If the method is used for labeling a substrate in vivo, the method may be carried out in the absence of the methionine adenosyl transferase (MAT) to avoid a competition with unlabelled S-adenosyl methionine (SAM), which is generated by native S-adenosyl methionine (SAM) production in the host cell. Thus, the host cell used may be methionine adenosyl transferase (MAT) deficient. It may be further required express the S-adenosyl transporter in the host cell, which is methionine adenosyl transferase (MAT) deficient, to ensure viability of the host cell. It may also be required to overexpress the nucleoside transporter and NupC and/or NupG to increase the intracellular adenosine concentration. It may further be required to overexpress the genes for MetA, MetB and MetC to enable production of homocysteine. Finally, it may also be required to overexpress adenosylhomocysteine hydrolase (EC 3.3.11 ) to enable production of SAH from homocysteine and adenosine.
After production of the alkylated carrier compound, the alkylated carrier compound may be isolated and optionally purified. The alkylated carrier compound may be isolated from the cell-free system or buffer if it was produced in vitro. Alternatively, the alkylated carrier compound may be isolated from a ceil if it was produced in vivo. The cells may be lysed by any technique known to the skilled person, and the alkylated carrier compound is purified from the supernatant after removing ceil debris by centrifugation.
Kit for producing an alkylated carrier compound
The present invention further relates to a kit for producing an alkylated sulfur- or selenium- containing carrier compound comprising a S-alkyltransferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above. The S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase comprised in the kit for producing an alkylated sulfur- or selenium-containing carrier compound.
The alkylated sulfur- or selenium-containing carrier compound may be isolated and optionally purified.
The obtained alkylated sulfur- or selenium-containing carrier compound is preferably as defined herein above. In a preferred embodiment a methylated sulfur- or selenium-containing carrier compound is produced. In this embodiment, the optionally substituted alkyl group is preferably a methyl group. It is preferred that the alkylated sulfur- or selenium-containing carrier compound produced with the kit is a methylated sulfur-containing carrier compound, more preferably S-adenosyl-methionine (SAM). The sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
The S-alkyltransferase, which is preferably comprised in the kit for producing S-adenosyl- methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound, is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum. If a halide methyl transferase is comprised, the alkyl donor comprised in the kit may be methyl iodide, methyl bromide or methyl chloride, preferably methyl iodide or methyl chloride, more preferably methyl iodide.
It is also preferred that a methionine S-methyltransferase (MSMT), preferably a methionine
S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp. JA431, as S- alkyltransferase is comprised in the kit for producing S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound. If a methionine S- methyltransferase (MSMT) is comprised in the kit, the alkyl donor comprised is preferably S- methyl methionine or Se-methyl selenomethionine.
Most preferably the kit is for producing S-adenosyt-methionine (SAM), wherein the kit comprises S-adenosyl homocysteine <SAH) as carrier compound prior to alkylation, a halide methyl transferase as S-alkyltransferase and methyl iodide as alkyl donor. Also preferred is a kit for producing S-adenosyl-methionine (SAM), wherein the kit comprises S-adenosyl homocysteine (SAH) as carrier compound prior to alkylation, a methionine S- methyltransferase as S-alkyltransferase and S-methyl methionine as alkyl donor.
The kit for producing an alkylated sulfur- or selenium-containing carrier compound, is preferably substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC
3.2.2.9). The term substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC
3.2.2.9) is understood as defined above. The absence of the S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9) is preferred for the reasons explained herein above.
Use for the production of an alkylated carrier compound
The present invention also relates to the use of a S-alkyltransferase as defined herein above, an alkyl donor as defined herein above and a sulfur- or selenium-containing carrier compound as defined herein above for the production of an alkylated carrier compound. The S-alkyltransferase is an alkyltransferase as used in step a) of the alkylation method in accordance with the invention. Thus, all definitions for the alkyltransferase used in step a) also relate to the S-alkyltransferase used for the production of an alkylated substrate.
The present invention further relates to the use of a kit as defined herein above for the production of an alkylated carrier compound.
The optionally substituted alkyl group is preferably a methyl group. It is preferred that the alkylated sulfur- or selenium-containing carrier compound produced is a methylated sulfur- containing carrier compound, more preferably S-adenosyl-methionine (SAM). The sulfur- or selenium-containing carrier compound is preferably S-adenosyl homocysteine (SAH).
The S-alkyitransferase, which is preferably used for the production of S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound, is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum. If a halide methyl transferase is used, the alkyl donor is preferably selected from methyl iodide, methyl bromide and methyl chloride, more preferably methyl iodide or methyl chloride, still more preferably methyl iodide.
It is also preferred that a methionine S-methyltransferase (MSMT), preferably a methionine
S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp. JA431, as S- alkyltransferase is used for the production of S-adenosyl-methionine (SAM) as alkylated sulfur- or selenium-containing carrier compound. If a methionine S-methyltransferase (MSMT) is used, the alkyl donor comprised is preferably S-methyl methionine or Se-methyl selenomethionine.
Most preferably S-adenosyl-methionine (SAM) is produced using S-adenosyl homocysteine (SAH) as carrier compound prior to alkylation, a halide methyl transferase as S- alkyltransferase and methyl iodide as alkyl donor. Also preferred for the production of S- adenosyl-methionine (SAM) is the use of S-adenosyl homocysteine (SAH) as carrier compound prior to alkylation, a methionine S-methyltransferase as S-alkyltransferase and S- methyl methionine as alkyl donor.
The alkylated carrier compound may be produced in vitro or in vivo. Any limitations described above for the method for producing an alkylated carrier compound also apply to the use for the production of an alkylated carrier compound.
The alkylated substrate is preferably produced in the absence of S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9). After production of the alkylated carrier compound, the alkylated carrier compound may be isolated and optionally purified. The alkylated carrier compound may be isolated from the cell-free system or buffer if it was produced in vitro. Alternatively, the alkylated carrier compound may be isolated from a cell if it was produced in vivo. The cells may be lysed by any technique known to the skilled person, and the alkylated carrier compound is purified from the supernatant after removing cell debris by centrifugation.
In the Examples, it was demonstrated that, surprisingly, an alkyl carrier compound can be efficiently (re-)generated using the inventive system.
In particular, Example 1 demonstrates that several methyltransferases with N-, C- or O- specificity (EgtD, IMT; PMT, SgvM and NovO), which catalyze methylation of a variety of specific substrates using S-adenosyl methionine (SAM) as co-substrate, can be combined with a S-specific halide methyitransferase and methyl iodide as stoichiometric alkyl donor to regenerate the catalytic methyl carrier S-adenosyl methionine (SAM). Example 1 demonstrates that the methyitransferase for alkylating a substrate and its substrates are not limiting for the system. Thus, it can be assumed that the system is applicable to any alkyltransferase-substrate combination.
Example 1 further demonstrates that S-adenosyl methionine (SAM) can be generated, i.e. produced, from S-adenosyl homocysteine (SAH) using the S-specific halide methyitransferase and methyl iodide as alkyl donor.
Example 1 additionally shows that the (re-Jgeneration of the carrier compound S-adenosyl methionine (SAM) can be further improved by minimizing the contamination with S-adenosyl homocysteine nucleosidase. S-adenosyl homocysteine nucleosidase is a major source of S- adenosyl homocysteine degradation. Removal of S-adenosyl homocysteine nucleosidase can be achieved either by re-purifying the employed alkyltransferase or by deleting the gene encoding the S-adenosyl homocysteine nucleosidase in the host cell, which is used for expressing the employed alkyltransferases.
Example 2 shows that the system is not limited to a halide methyitransferase as S- alkyitransferase and methyl iodide as alkyl donor. It is documented in Example 2 that the S- specific methyitransferase methionine S-methyltransferase can be combined with S-methyl methionine as alkyl donor to (regenerate the spent alkyl carrier S-adenosyl homocysteine (SAH) to S-adenosyl methionine (SAM). In Example 2 the alkylation of the substrate is catalyzed by the N-specifc methyitransferase OvoC in a SAM-dependent manner.
Example 1 further demonstrates that S-adenosyl methionine (SAM) can be generated, i.e. produced, from S-adenosyl homocysteine (SAH) by using the S-specific methionine S- methyltransferase and S-methyl methionine as alkyl donor.
Accordingly, the appended examples plausibly demonstrate that an S-aikyltransferase and an alkyl donor can be used to generate S-adenosyl methionine (SAM) from S-adenosyl homocysteine (SAH). It can be expected that this system is also applicable to other alkyl carrier compounds, for example selenium-based SAH/SAM derivatives or methionine-based alkyl carrier compounds. It has been further plausibly demonstrated that the S- alkyltransferase, alkyl donor can be employed in the alkylation of a substrate by a further alkyltransferase to regenerate the co-substrate of the second alkyltransferase, i.e. the alkyl carrier compound.
Consequently, the experimental data in the Examples provide a clear rationale to use the methods, the compositions and kits of the invention for alkylating a substrate and/or for producing an alkyl carrier compound.
There is no indication in the prior art that a simple mechanism which requires only a single enzymatic step for the ( regeneration of a co-substrate of alkyltransferases can be used to provide an efficient alkylation method . The prior art method are either based on chemical synthesis of the co-substrate or involve complicated enzyme cascades. Accordingly, the system as plausibly exemplified herein below, is surprising.
The invention as described above may find application e.g. in the following contexts:
A) Preparative methyl transfer biocatalysis: Application of SAM-dependent enzymes for cell-free chemical transformations has been prohibited by the large costs of SAM. The inventors demonstrated that the HMT-catalyzed SAM-regeneration system (HCSR system) can be combined with N-, O- and C-methylating m ethyltra nsfe ras es . Based on these findings, it can be anticipated than any SAM-dependent methyl transferase with the ability to transfer methyl groups or other alkyl groups from SAM or SAM-derivatives to a substrate may be combined with the HCSR system to generate preparative methylation reaction systems.
Applications include: a) preparative methylation of natural products or synthetic compounds; b) production of isotopicaily labelled natural products or synthetic compounds for the use as fine chemicals, radiopharmaceuticals and theranostics. The following methyltransferases are involved in the biosynthesis of biomedically relevant compounds and thus of particular interest: the carminomycin 4-O-methyitransferase DnrK, the demethylrebeccamycin-D- glycose O-methyltransferase RebM, the S-adenosyl-L-methionine-dependent O- methyltransferase SynOMT, the O-m eth yltra n sferase BcOMT2, the 2,7-dihydrocy-5-methyl- 1 -naphthoate 7-O-methyltransferase NcsB1 , the phosphonate O-methyltransferase Dhpl, the demethyldecarbamoylnovobiocin O-methyltransferase NovP, the methyltransferase CaiOI , O-methyltransferase Cal06, the mitomycin 6-O-methyltransferase MmCR, the mycinamicin VI 2”-0-methyltransferase MycE, 10-hydroxycamptothecin O-methyltransferase, the Norbelladine 49-O-Methyltransferase, the phenazine-1 -carboxylate N-methyltransferase PhzM, the dTDP-3-amino-3,4,6-trideoxy-alpha-D-glycopyranose N,N-dimethyltransferase DesVI, the NodS N-methyltransferase, the dTDP-3-amino-3,6-dideoxy-alpha-D- glucopyranose N , N-d imethy Itransferase TylM1 , the Indolethylamine N-methyltransferase, Phenylethanolamine N-methyltransferase, beta-alanine N-methyltransferase, dimethylglycine methyltransferase, Pavine N-Methyltransferase, Psilocybin synthase PsiM, Dimethylailyltryptophan N-methyltransferase, Reticuline N-Methyltransferase, Picrinine N- Methyltransferase, Norajmaline N-methyltransferase, (S)-coc!aurine-N-methyltransferase, the T etrahydroprotoberberine cis-N-Methyltransferase, the cyclopropane-fatty-acyl-phospholipid synthase CPFAS, the methylene-fatty-acyl-phospholipid synthase, the uroporphyrinogen-lll C-methyltransferase UMT, the sterol C-methyltransferase sterol 24-C-methyltransferase, the gamma-tocopherol 5-O-methyltransferase.
B) Biocatalytic production of SAM and SAM derivatives: SAM is a very expensive reagent. The results described above show that HMTs can catalyze stereoselective alkylation of SAH to form SAM using methyl iodide as methyl donor. SAH can be synthesized in large scale by chemical synthesis (Figure 30). SAM could be isolated by simple ion- exchange chromatography. The same technology is also amenable to the production of SAM derivatives using SAH and alternative alkyldonors (for example: ethyl iodide, propyl iodide, propargyl iodide).
C) Chemoenzymatic production of SAM. This widely used reagent is expensive and the commercial products usually consist of ill-defined mixtures of the (S,S)- and (R,S) diastereomers of SAM. The inventors found that HMT-cataiyzed methylation of SAH produces the active (S,S) isomer of SAM in pure form. Chemical synthesis of SAH is straightforward, suggesting that SAM can be produced commercially using the HMT methylation technology.
D) Chemoenzymatic production of natural product derivatives. The Braunschweig Enzyme database lists more than 300 MTs (EC 2.1.1.-) with different substrate specificities. The SAM-regeneration system described above should be applicable to all of these enzymes and provide a general solution for cell-free applications of MTs to large-scale applications. C~ methylating enzymes are particularly interesting because of their ability to form asymmetric C-C bonds. In addition, methyltransferase (MT) catalysis is not limited to methyl transfers. The HMT/MT technology will also allow catalytic transfer of fluoromethyl groups, stable isotope lables H-methyl, 13C-methyl), or larger alkyl chains (ethyl, propyl, propargyl).
E) PET tracer synthesis. High substrate specificity combined with high catalytic efficiency also predestinates MT catalysis for applications in time-sensitive synthesis. An important example is the synthesis of 11C and 1SF labeled biomolecules. Both radioisotopes are short lived (t1/2 < 2h) and decay by emission of a positron. Positron emission tomography (PET) has emerged as an important imaging technique to diagnose disease and to examine drug pharmacokinetics. Reagents such [11C]-methyl iodide or [18F]-ftuoromethyl iodide are common reagents for chemical synthesis of 11C or 18F-labeled amino acids or carbohydrates. The HMT/MT technology provides an entirely new perspective for Positronen-Emissions- Tomographie (PET) tracer synthesis.
F) Bionic pathways for in vivo methylation
Further applications include: i) Utilization of artificial methyl donors for SAM-dependent methylation in growing cells with a disrupted C1 -metabolism.
ii) Utilization of methyl donors other than methyl halides: S-methyl methionine or methionine.
iii) Utilization of HMT/MT catalysed SAM regeneration to support methylation reactions catalysed by SAM-radical methyltransferases.
System 1: Whole-cell formulation of the HMT/MT cascade. HMT/MT proteins could be overexpressed in E. coli (or other industrial microorganisms, such as C. glutamicum). After growth of biomass is completed, cells are perforated and washed to eliminate small molecules but retain proteins, and nucleic acids. This treated biomass is added to solutions of substrate, methyl donor (methyl halide) and catalytic amounts of SAH. Completed reactions are filtered and lyophilized to produce near homogeneous salts of product. This system is particularly useful for transfer reactions of methyl- and other alkyl groups, and stable isotopologues thereof. System 2: Decoupling C1 -metabolism from the SAM cycle. The HMT/MT could also allow specific attachment of labelled methyl-groups (13C, 2H) onto substrates in living cells. Cells, which can tolerate high concentrations of methyl halides (1 - 10 mM), could regenerate SAH using HMT to form labelled SAM, which is then used for specific product methylation by an overproduced MT. To avoid competition by unlabelled SAM emerging from the native SAM cycle (i.e production of SAM from ATP and methionine), it may be required to render the production strain unable to make SAM any other way. This car» be achieved by deleting methionine adenosyl transferase (MAT, EC 2.5.1 6), Instead the cell can make SAH from homocysteine and adenosine catalysed by adenosylhomocysteine hydrolase (EC 3.3.11), The thermodynamic equilibrium of this reaction favors the synthesis of SAH, SAH is then methylated by HMT, In order to stabilize SAH the strain may also need to be deficient of SAH nucleosidase (EC 3 2.2.9), The obtained celt should be able to produce SAM from methyl chloride, adenosine and homocysteine. Cells fed with isototopica!ly labelled methyl halides will be able to produce methylated products with high retention of the isotopic label, while avoiding isotope insertion in SAM-independent biosynthetic pathways,
System 3: In vivo isotope labelling using methionine or S-methyl methionine as methyl donor. Labelled S-methyl methionine (SMM) is synthesized and fed to cells (e.g. E coir). One methyl group of this metabolite is transferred to homocysteine by the enzyme S- methylmethionine-homocysteine S-methyltransferase (EC 2.1.1.10). Both methionine equivalents are activated to SAM by methionine adenosyl transferases (MAT, EC 2.5.16) and used for substrate methylation by an overproduced MT. The engineered cells with no other source of methionine (i.e, cells deficient in EC 2.11.5, EC:2. 14, EC:2 1113) could only make labelled methionine and labelled SAM, ensuring efficient and complete labelling of products. Genetic deletion of these methionine biosynthetic genes may not be necessary due to metabolic downregulation by the availability of exogeneous sources of methionine.
The obtained cell may be fed with isototopicaliy labelled methionine or S-methyl methionine to produce methylated products with high retention of the isotopic label, while avoiding isotope insertion in SAM-independent biosynthetic pathways (similar to System 2). This system does not require methyl halides as methyl donors. This system could be of particular interest for the insertion of fluoromethyl-groups (using S-fluoromethyl methionine as methyl donor).
System 4: Application of the HMT/MT technology to SAM-radical methy Itra nsferases.
SAM-radical methyltransferases (radical-MTs) represent a large class of methyltransferases that can catalyse difficult methyiation reactions, HMT mediated SAM regeneration could also support radical MTs catalysed reactions.
In vitro applications include: Cascades containing HMT and radica!-MTs could be constructed with few adaptation of the protocol described for HMT/MT cascades. Numerous radical MTs consume two equivalents of SAM per methyl transfer. One equivalent is converted to SAH, the second equivalent is converted to methionine and 5-deoxy adenosine (5dAdo). Therefore, SAM needs to be regenerated by two different systems: i) SAH could be recycled by HMT and methyl halide ii) Methionine could be recycled by adenosyl-chloride synthase (chlorinase) using the (synthetic) cosubstrate 5-chloro-5-deoxy adenosine (5-CI- dAdo). Overall this cascade consists of three enzymes: HMT, radical-MT and chlorinase. The Overall reaction consumes methyl halide, 5-Cl-dAdo and substrate. The reaction produces methylated product, chloride and SdAdo,
In vitro applications include: A simitar approach as described in system 2 (i.e, decoupling the C1 -metabolism from the SAM cycle) could be implemented to design production strains that introduce labelled methyl groups into specific products,
Radical-MTs catalyse very difficult chemical reactions. Exploitation of such enzymes for biocatalytic application will significantly broaden the scope of enzyme-based synthesis. This cascade provides a methodology to use radicai-MTs without the need for SAM as a stoichiometric reagent. Instead, the reaction is driven by the relatively simple reagents methyl halide and 5-Cl-dAdo.
The invention relates to the embodiments as defined in the claims and in the following items as also herein described above and below:
1 , A method for alkylating a substrate
which comprises the following steps:
a) an aikyiation step wherein a S-alkyitransferase transfers an optionally substituted alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound;
b) an alkylation step wherein a N-, G-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound, and wherein at least a part of the dealkylated sulfur- or selenium-containing carrier compound yielded in step b) is recycled to step a) to regenerate the alkylated sulfur- or selenium-containing carrier compound
2. The method according to item 1 , wherein step a) is carried out as a first alkylation step.
3. The method according to item 1 or 2, wherein each sulfur- or selenium containing carrier compound molecule is subjected to the alkylation step a) on the average at least 2 times, more preferably at least 5 times.
4. The method according to any of items 1 to 3, wherein, in step a), a S-alkyltransferase transfers the optionally substituted alkyl group from the alkyl donor to a sulfur- containing carrier compound, yielding an alkylated sulfur-containing carrier compound; and
in alkylation step b), a N-, C-, 0-, S-, or P-alkyitransferase transfers the optionally substituted alkyl group from the alkylated sulfur-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur-containing carrier compound,
and wherein at least a part of the dealkylated sulfur-containing carrier compound yielded in step b) is recycled to step a).
5. The method according to stem 4, wherein the sulfur-containing carrier compound prior to alkylation comprises a thioether moiety,
6. The method according to item 4 or 5, wherein the sulfur-containing carrier compound prior to alkylation is represented by the following formula (la):
Figure imgf000057_0001
wherein,
R1 is selected from a group consisting of a carboxy group, a hydrogen, a triazole, and an ester group;
R2 is selected from a group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy group;
R3 and R4 are independently hydrogen or a hydroxy group, preferably a hydroxy group; and
B1 is an optionally substituted purine or thienoadenine. The method according to any of items 4 to 6, wherein the sulfur-containing carrier compound prior to alkylation is selected from the group consisting of S-adenosyl homocysteine (SAH), 2-fluoroadenosy! homocysteine, S-(5'-adenosyl)-3- thiopropylamine, N6substituted S-adenosyl homocysteine, S-adenosyl N- acetylhomocysteine, S-adenosyl N , N ,-dimethylhomocysteine, S-adenosyl homocysteine methylester, S-adenosyl homocysteine ethylester and S-adenosyl homocysteine phenylester, The method according to any of items 4 to 7, wherein the sulfur-containing carrier compound prior to alkylation is S-adenosyl homocysteine (SAH). The method according to item 4, wherein the sulfur-containing carrier compound prior to alkylation is represented by the following formula (lb);
Figure imgf000058_0001
wherein R5 is selected from the group consisting of a methyl group which may be substituted, an ethyl group which may be substituted and a propyl group which may be substituted. . The method according to item 4 or 9, wherein the sulfur-containing carrier compound prior to alkylation is selected from the group consisting of methionine, monofluoromethyl homocysteine, difluoromethyl homocysteine and trif!uoromethyi homocysteine. , The method according to any of items 1 to 3, wherein, in step a), a S-alkyltransferase transfers the optionally substituted alkyl group from the alkyl donor to a selenium- containing carrier compound, yielding an alkylated selenium -containing carrier compound; and in alkylation step b), a N-, C-, 0-, S-, or P-aikyltransferase transfers the optionally substituted alkyl group from the alkylated selenium -containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated selenium -containing carrier compound,
and wherein at least a part of the dealkylated selenium -containing carrier compound yielded in step b) is recycled to step a)
12, The method according to item 11 , wherein the selenium-containing carrier compound prior to alkylation comprises a selenoether moiety,
13. The method according to item 11 or 12, wherein the selenium-containing carrier compound prior to alkylation is represented by the following formula (lc):
Figure imgf000059_0001
wherein,
R6 is selected from a group consisting of a carboxy group, a hydrogen, a triazole, and an ester;
R7 is selected from a group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy;
R8 and R9 are independently hydrogen or a hydroxy group, preferably a hydroxy group; and
B2 is an optionally substituted purine or thienoadenine.
14. The method according to any of items 1 1 to 13, wherein the selenium-containing carrier compound prior to alkylation is selected from the group consisting of Se-adenosyi se!enohomocysteine, thienoadenosyl selenohomocysteine, Se-( 2-f luoroadenosyl ) selenohomocysteine, Se-(5'-adenosyl)-3-selenopropylamine, Nd-substituted Se- adenosyl selenohomocysteine, Se-adenosyl N-acetyt selenohomocysteine, Se- adenosyl N,N, -dimethyl selenohomocysteine, Se-adenosyl selenohomocysteine methylester, Se-adenosy! selenohomocysteine ethylester and Se-adenosyl selenohomocysteine phenylester, preferably Se-adenosyl selenohomocysteine. 15 The method according to any of items 1 to 14» wherein the alkyltransferase of step b) is a N-» C-, 0-, or P-alkyltransferase, preferably a N-» C-, or O-alkyltransferase.
18. The method according to any of items 1 to 15» wherein the alkyltransferase used in step a) is an S-adenosyl methionine (SAM) dependent alkyltransferase.
17. The method according to any of items 1 to 16» wherein the alkyltransferase used in step b) is an S-adenosyl methionine (SAM) dependent alkyltransferase.
18. The method according to any of items 1 to 8 and 11 to 17» wherein the method is carried out in the absence of S-adenosyl homocysteine (SAH) nucleosidase.
19. The method according to any of items 1 to 18» wherein the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT), thioether methyltransferases and N-methyl proline methyltransferase, N-dimethyl glycine methyltransferase and N-dimethyl beta-alanine N-methyltransferase, wherein the thioether methyltransferase is preferably selected from methionine 8- methyltransferase (MSMT)
20. The method according to any of items 1 to 19, wherein the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT) and methionine S-methyltransferase (MSMT).
21. The method according to any of items 1 to 18» wherein the alkyltransferase used in step a) is a ca rboxyl m eth yltransferase , preferably a indole-3-acetic acid carboxyl methyltransferase, Trigonelline synthase, Gibberellic acid methyltransferase, salicylic acid carboxyl methyltransferase, Jasmonic acid carboxyl methyltransferase, or Caffeine synthase.
22. The method according to item 19 or 20, wherein the halide methyl transferase (HMT) originates from bacteria, plants, fungi or archaea.
23. The method according to item 22, wherein the halide methyl transferase originates from an acidobacterium, more preferably from chloracidobacterium thermophilum. 24. The method according to item 19 or 20, wherein the methionine S-methyltransferase (MSfvJT) originates from bacteria, plants, fungi or archaea.
25. The method according to item 24, wherein the methionine S-methyltransferase (MSMT) originates from a rhodobacter, more preferably from Rhodobacter sp. JA431
26. The method according to any of items 1 to 25, wherein the alkyltransferase used in step b) transfers the optionally substituted alkyl group by a SN2-type reaction,
27. The method according to any of items 1 to 26, wherein the alkyltransferase used in step b) is selected from the group consisting of
the carminomycin 4-O-methyltransferase DnrK, the demethylrebeccamycin-D-glycose O-methyltransferase Rebfvt, the S-adenosyl-L-methionine-dependent O- methyltransferase SynOMT, the O-methyltransferase BcOMT2, the 2,7-dihydrocy-5- methyl-1 -naphthoate 7-O-methyltransferase NcsB1 , the phosphonate O- methyltransferase Dhpl, the demethyldecarbamoylnovobiocin O-methyltransferase NovP, the methyltransferase CalOI , O-methyltransferase Cal06 the mitomycin 6-0- methyltransferase MmCR, the mycinamicin VI 2"-0-methyltransferase MycE, 10- hydroxycamptothecin O-methyltransferase, the Norbelladine 49-O-Methyltransferase, the phenazine-1 -carboxylate N-methyltransferase PhzM, the dTDP-3-amino-3,4,6- trideoxy-alpha-D-glycopyranose , N-d i methyltransfera se DesVl, the NodS N- methyltransferase, the dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N- d i m ethyltra n sf era se TylM1 , the Indolethylamine N-methyltransferase,
Phenylethanolamine N -m ethyltran sf erase , beta-alanine N-methyltransferase, dimethylglycine methyltransferase, Pavine N-Methyltransferase, Psilocybin synthase PsiM, Dimethyiallyltryptophan N-methyltransferase, Reticuline N-Methyltransferase, Picrinine N-Methyltransferase, Norajmaline N-methyltransferase, (S)-coclaurine-N- methyltransferase, the Tetrahydroprotoberberine cis-N-Methyltransferase, the cyclopropane-fatty-acyl-phospholipid synthase CPFAS, the methylene-fatty-acyl- phospholipid synthase, the uroporphyrtnogen-lll C-methyltransferase UMT, the sterol C-methyltransferase sterol 24-C-methyltransferase, the gamma-tocopherol 5-0- methyltransferase,
28. The method according to any of items 1 to 27, wherein the the alkyltransferase used in step b) is selected from the group consisting of the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase (IMT), the putrescine N-methyl transferase (PMT), the methyltransferase SgvM, the 8- demethylnovobiocic acid C8-methyltransferase NovO and the 5-thiohistidine N- methyltransferase OvoC.
29. The method according to any of items 1 to 25, wherein the alkyltransferase used in step b) transfers the optionally substituted alkyl group by a radical reaction.
30. The method according to any of items 1 to 25 and 29, wherein the alkyltransferase used in step b) is selected from the group consisting of Tryptophan 2-C- methyltransferase TsrM, P-methyltransferase, Valine methyltransferases PoyC, CI0N6, Pyrrole-2-carboxyl methyltransferase, Fosfomycin Biosynthesis Enzyme Fom3, GenK, Gentamicin biosynthetic methyltransferase.
31. The method according to any of items 1 to 30, wherein the method is carried out in vitro, preferably in a cell free-system or a buffer.
32. The method according to any of items 1 to 31 , wherein the method is carried out in vivo.
33. The method according to any of items 1 to 32, wherein the sulfur- or selenium- containing carrier compound is present in an amount which is substoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate.
34. The method according to any of items 1 to 33, wherein the alkyl donor is present in an amount which is at least stoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate.
35. The method according to any of items 1 to 34, wherein the alkyl donor is a compound comprising the optionally substituted alkyl group and a leaving group.
36. The method according to any of items 1 to 35, wherein the alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group, preferably a methyl group.
37. The method according to any of items 1 to 36, wherein the optional substituent is selected from the group consisting of an alkenyl group, an alkinyl group, and a fluorine atom,
38. The method according to any of items 1 to 37, wherein the optionally substituted alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyl group, a difluoromethyl group and a trifluoromethyl group, preferably a methyl group.
39. The method according to any of items 1 to 38, wherein the optionally substituted alkyl group is isotopically labeled.
40. The method according to any of items 35 to 39, wherein the leaving group is selected from the group consisting of iodide, bromide, chloride, methionine, selenomethionine, sulfate, dimethy!sulfoxide and a cyclic or linear thioether, preferably iodide, bromide and chloride, more preferably iodide and chloride, even more preferably iodide.
41. The method according to any of items 1 to 40, wherein the alkyl donor is selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, propargyl bromide, propargyl chloride, S-m ethyl methionine, Se-methyl selenomethionine, methyl sulfate, 8-methyl tetrahydrothiophene, S- methyl thietane, S- methyl ethylene sulphide, S-methyl di(monofluoromethyl) sulphide, S-methyl dimethyl sulphide, 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid, S-methyl 3- (methylthio)propionic acid, and trimethylsulfoxonium iodide,
preferably selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, S-methyl methionine and Se-methyl selenomethionine.
42. The method according to any of items 1 to 41 , wherein the alkyl donor is selected from the group consisting of methyl iodide, methyl bromide, methyl chloride and S-methvl methionine, preferably methyl iodide, methyl bromide and methyl chloride, more preferably methyl iodide and methyl chloride, even more preferably methyl iodide.
43. The method according to any of items 1 to 34, wherein the alkyl donor is represented by the following formula (II):
Figure imgf000064_0001
wherein
R10 is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyt group, a butyl group, a monof!uoromethyl group, a difluoromethyl group and a trifluoromethyl group, preferably a methyl group;
X is selected from the group consisting of NH, O and S; and
R10 is selected from an alkyl group or an aryl group, preferably a methyl group or ethyl group,
44. The method according to any of items 1 to 43, wherein the substrate comprises a nucleophilic atom selected from the group consisting of a nitrogen atom, a carbon atom, an oxygen atom, a sulfur atom and a phosphorus atom
45. The method according to any of items 1 to 44, wherein the substrate comprises
a OH acidic carbon-hydrogen bond; or
a group selected from the group consisting of a primary amino group, a secondary amino groups, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group and phosphine group,
preferably primary amino group, a secondary amino groups, a hydroxy group, an amide group, a carboxy group.
46. The method according to any of items 1 to 45, wherein the substrate is selected from the group consisting of a small molecule with a molecular mass of less than 1 kDa, primary metabolites, secondary metabolites, peptides such as ribosomal peptides, proteins, polysaccharides and nucleic acids,
wherein the primary metabolite is preferably selected from the group consisting of amino acids, nucleosides, nucleotides, lipids, carbohydrates and/or
the secondary metabolites are preferably selected from the group consisting of non ribosomal peptides, polyketides, terpenes, alkaloids, phenylpropanoids, purine and pyrimidine based metabolites.
47. The method according to any of items 29 to 46, wherein the alkyltransferase used in step b) is a SAM-dependent alkyltransferase, preferably a SAM-dependent methyltransferase, and wherein the sulfur-containing carrier compound is S-adenosyl homocysteine (SAH), and wherein step b) additional yields methionine
48. The method according to item 47, wherein at feast a part of the methionine yielded in step b) is regenerated by an adenosyl-chloride synthase, which synthesizes S- adenosyl-L-methionine (SAM) from methionine and a co-substrate.
49. The method according to item 48, wherein the co-substrate is 5-chloro-5-deoxy adenosine.
50. A kit for alkylating a substrate comprising a S-alkyltransferase as defined in any of items 1 , 16 and 19 to 25, a N-, C-, 0-, S-, or P-alkyltransferase as defined in any of items 1 , 15, 17, 26 to 30 and 47 to 49, an alkyl donor as defined in any of items 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of items 1 and 3 to 14.
51. The kit according to item 50, further comprising a substrate as defined in any of items 44 to 46.
52. The kit according to any of items 50 or 51 , which is substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9).
53. The kit according to any of items 50 to 52, further comprising an adenosyl-chloride synthase and a co-substrate, which is preferably 5-ch!oro-5-deoxy adenosine.
54. Use of a S-alkyltransferase as defined in any of items 1 , 16 and 19 to 25, a N-, C-, 0-,
S-, or P-alkyltransferase as defined in any of items 1 , 15, 17, 26 to 30 and 47 to 49, an alkyl donor as defined in any of items 1 and 35 to 43 and a sulfur- or selenium- containing carrier compound as defined in any of items 1 and 3 to 14 or a kit as defined in any of items 50 to 53 for the production of an alkylated substrate.
55. The use according to item 54 for the production of an alkylated substrate, wherein the substrate prior to alkylation or the alkylated substrate is as defined in any of items 44 to 46.
56. The use according to item 54 or 55 for the production of an alkylated substrate, wherein the alkylated substrate is isotopically labeled. 57. The use according to any of items 54 to 56 for the production of an alkylated substrate in vitro, preferably in a cell-free system or a buffer.
58. The use according to any of items 54 to 57 for the production of an alkylated substrate in vivo.
59. The use according to any of items 54 to 57 and 58 for the production of an alkylated substrate in a bacterial cell, plant cell or fungal cell.
60. The use according to any of items 57 to 57 and 58 to 59 for the production of an alkylated substrate in a bacterial cell, wherein the bacterial cell is preferably E.coii or C. glutamicum.
61. The use according to any of items 57 to 60 for the production of an alkylated substrate, wherein the alkylated substrate is isolated from a cell.
62. A method for producing an alkylated sulfur- or selenium-containing carrier compound, comprising an alkylation step, wherein a S-alkyltransferase as defined in any of items 1 , 16 and 19 to 25 transfers an optionally substituted alkyl group as defined in any of items 1 , 36 to 39 from an alkyl donor as defined in any of items 1 and 35 to 43 to a sulfur- or selenium-containing carrier compound as defined in any of items 1 and 3 to 14, yielding an alkylated sulfur- or selenium-containing carrier compound.
63. The method according to item 62, wherein the method is carried out in vitro, preferably in a cell-free system or a buffer.
64. The method according to item 62 or 63, wherein the alkylated sulfur- or selenium- containing carrier compound is isolated and optionally purified.
65. The method according to any of items 62 to 64, wherein the optionally substituted afkyl group is a methyl group.
66. The method according to any of items 62 to 65, wherein the sulfur- or selenium- containing carrier compound is a sulfur-containing carrier compound, preferably S- adenosyl homocysteine (SAH) and the alkylated sulfur- or selenium-containing carrier compound is a methylated sulfur-containing carrier compound, preferably S-adenosyl- methionine (SAM),
67. The method according to any of items 62 to 66, wherein the method is carried out in the absence of S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2, 2 9).
68. The method according to any of items 62 to 67, wherein the S-alkyltransferase is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum.
69. The method according to any of items 62 to 68, wherein the alkyl donor is methyl iodide, methyl bromide or methyl chloride, preferably methyl iodide or methyl chloride, more preferably methyl iodide.
70. The method according to any of items 62 to 67, wherein the S-alkyltransferase is a methionine 5-methyltransferase (MSMT), preferably a methionine S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp JA431.
71. The method according to any of items 62 to 67 and 70, wherein the alkyl donor is S- methyl methionine.
72. A kit for producing an alkylated sulfur- or selenium-containing carrier compound comprising a S-alkyltransferase as defined in any of items 1 , 16 and 19 to 25, an alkyl donor as defined in any of items 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of items 1 and 3 to 14.
73. The kit according to item 72 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the sulfur- or selenium-containing carrier compound and the alkylated sulfur-containing carrier compound are as defined in item 66.
74. The kit according to item 72 or 73, which is substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9).
75. The kit according to any of items 72 to 74 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the S-alkyltransferase is as defined in item 68,
76. The kit according to any items 72 to 75 for producing an alkylated sulfur- or selenium- containing carrier compound, wherein the alkyl donor is as defined in item 69.
77. The kit according to any of items 72 to 74 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the S-alky!transferase is as defined in item 70.
78. The kit according to any items 72 to 74 and 77 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the alkyl donor is as defined in item 71.
79. Use of a S-a!kyltransferase as defined in any of items 1 , 16 and 19 to 25, an alkyl donor as defined in any of items 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of items 1 and 3 to 14 or a kit as defined in any of items 72 to 78 for the production of an alkylated carrier compound.
80. The use according to item 79, for the production of an alkylated carrier compound in vitro, preferably in a cell-free system or a buffer.
81. The use according to item 79 or 80, wherein the alkylated sulfur- or selenium- containing carrier compound is isolated and optionally purified.
82. The use according to any of items 79 to 81 , wherein the sulfur- or selenium-containing carrier compound and the alkylated sulfur- or selenium-containing carrier compound are as defined in item 66.
83. The use according to any of items 79 to 82, wherein the S-alkyltransferase is as defined in item 68.
84. The use according to any of items 79 to 83, wherein the alkyl donor is as defined in item 69.
85. The use according to any of items 79 to 82, wherein the S-alkyltransferase is as defined in item 68. The use according to any of items 79 to 82 and 84, wherein the alkyl donor is as defined in item 69.
The present invention is further described by reference to the following non-limiting figures and examples, The Figures show:
Figure 1 : MT/HMT cascade for preparative methylation. Faint: alternative scheme for cyclic regeneration of SAM described in (Mordhorst, S., Siegrist, J., Mueller, M„ Richter, M., and Andexter, J N, (2017) Angew Chem Int Ed Engl. 56, 4037 - 4041 ), SAM-dependent methyltransferases (MTs) catalyze methyl transfers from SAM to a substrate forming methylated product/substrate and S-adenosylhomocysteine (SAH). A cyclic SAM regeneration system (faint) containing the enzymes adenosylhomocysteine hydrolase (SAHH), adenosine kinase (ADK), polyphosphate kinase 2 type I and If (PPK2-I, PPK2-II) and methionine adenosyltransferase (MAT) can recycle adenosine using methionine as methyl donor and polyphosphate as chemical energy (prior art). Halide methyl transferase (HMT) can directly remethylate SAH to SAM using methyl iodide as methyl donor (present invention). This reaction is driven by the high methyl transfer potential of methyl iodide as chemical energy.
Figure 2: HMT/EgtD methyl transfer cascade. HPLC-UV analysis monitoring HMT/EgtD mediated production of N-alpha-trimethyl histidine (TMH). Reaction composition: 1 mM histidine, 6 mM methyl iodide, 50 mM SAH, 10 mM HMT, 10 mM EgtD were run in 100 mM phosphate buffer at 25 °C. Time traces of reactions containing HMT/EgtD purified from BL21 cells; HMT/EgtD re-purified by size-exclusion chromatography. Proteins purified from a SAH nucleosidase-deficient E. coli strain.
Figure 3: Hydrolysis of SAH to adenine and ribosyl homocysteine.
Figure 4: SDS-PAGE of used enzymes. Left). HMT and EgtD expression in E. Coli BE21 (DE3) and purified by Ni' -affinity chromatography, lane 1. Marker with annotated MS (kD). lane 2. HMT, lane 3, EgtD. Middle). E1 MT and EgtD expression in E. Coli BE21(DE3) and purified by Ni"-affinity chromatography and size exclusion chromatography, lane 1. Marker with annotated MS. lane 2. HMT, lane 3, EgtD. Right). Enzymes expressed in new strain and purified by Ni"-affinity chromatography, lane 1. Marker with annotated MS, lane 2. HMT, lane 3. EgtD, lane 4. IMT, lane 5. SgvM, lane 6. PMT, lane 7, NovO.
Figure 5: Activity of HMT accepting SAM and iodide: Reactivity of HMT catalyzing methyl group transfer from SAM to iodide, a) Reaction scheme, b) HPLC trace of native reaction monitoring SAH and SAM 250 pL Reactions containing 100 mM sodium phosphate buffer (pH 8.0), 10 mM of iodide, 1 pM HMT and 1 mM SAM were incubated at 25 °C Figure 6: Activity of HMT accepting SAH and methyl iodide
Reactivity of HMT catalyzing methyl group transfer from methyl iodide to SAH. a) Reaction scheme, b) HPLC trace of reaction monitoring SAH consumption and SAM formation. 250 m!_ Reactions containing 100 mM sodium phosphate buffer (pH 8.0), 10 mM of methyl iodide, 1 mM HMT and 1 mM SAH were incubated at 25 °C.
Figure 7; Michaelis-Menten analysis of SAM formation catalyzed by HMT.
Michaeiis-Menten analysis of SAM formation catalyzed by HMT. 250 pL Reactions containing 100 mM sodium phosphate buffer (pH 8.0), 1 mM of methyl iodide, 0.5 mM HMT and different concentration of SAH were incubated at 25 °C. Concentration of SAH varies from 100 mM to 0.39 mM, 30 mΐ_ of reaction mixture was quenched by adding to 15 pL of 1 M phosphoric acid at 1 , 2, 3, and 4 minutes. Concentration of both SAH and SAM were monitored by Cation- exchange HPLC according to the standard curve obtained from solution of commercial SAH. The velocity were fitted to equation of v = vmax * [s] / (KM + [s]).
Figure 8: Identification of the degradation product
1H NMR of SAH degradation product and authentic adenine. Degradation product: 500 mΐ of 1 mM SAH and 40 mM EgtD in 100 mM sodium phosphate buffer was incubated overnight and lyophilized. The mixture was dissolved in 500 pL D20, a) Mixture of product and authentic adenine, b) Product of SAH degradation, c) Authentic adenine.
Figure 9: SAH degradation rate during HMT EgtD cascade reaction
Initial SAH degradation rate during HMT EgtD cascade reaction. Product was identified to be adenine. Therefore, concentration of degradation product was calculated based on the calibration curve of SAH. Concentrations of first four time points were fit versus time as linear curve. Slope of each curve were shown in figure as velocity, a) Proteins were purified from
BL21 by Ni" affinity chromatography, b). Proteins were re-purified by size-exclusion chromatography.
Figure 10: SAH nucleosidase activity of impurity within HMT from new strain
Reaction containing 1 mM of SAH and 10 mM of HMT purified from two strains were incubated at room temperature for 2 hours. No significant adenine was observed from the reaction of HMT purified from new strain after 2 hours. Figure 11 : Non-enzymatic SAH degradation rate.
Co-factor (i.e. carrier component) and its degradation during the cascade reaction using enzymes purified from new strain 500 mI_ reaction containing 1 mM histidine, 8 mM methyl iodide, 50 mM SAH, 10 mM HMT, 10 mM EgtD was run in 100 mM sodium phosphate buffer at 25 °C. a) Time curve of SAH, SAM and adenine, b) Adenine formation by non-enzymatic hydrolysis of SAH. Concentrations of adenine were fitted to linear curve and the slope was divided by concentration of SAH to get the reaction constant.
Figure 12: NMR assay monitoring the non-enzymatic methylation of S- adenosylhomocysteine
Non-enzymatic methylation of S-adenosylhomocysteine and buffer at pD 8. 600 mI_ of 100 mM phosphate 02O buffer (pD 8) containing 1 mM S-adenosylhomocysteine and 6 mM methyl iodide was incubated in NMR tube at room temperature (20°C). Another reaction without S-adenosylhomocysteine was run in parallel as control. 1H NMR was recorded at 30 minute, 20 h and 48 h. a) 1H NMR of methyl iodide solution in 100 mM phosphate D20 buffer after 48 h. b) 1H NMR of S-adenosylhomocysteine and methyl iodide after 30 minutes, c) S- adenosylhomocysteine and methyl iodide after 20 h. d) S-adenosylhomocysteine and methyl iodide after 48 h. e) Quantification of methylphosphate and methanol after 48 hours; Integral of methylphosphate is 0.3 (dH 3.33)14], which indicates the concentration is 100 mM. Integral of dimethylphosphate is 1.11 (dH 3.46)[4], which indicates the concentration of dimethylphosphate is 185 mM. Integral of methanol is 0.48 (dH 3.34}{5], which indicates the concentration is 160 mM.
Figure 13: Reaction rate after protein methylation
Protein methylation effect on activity of HMT-EgtD cascade reaction. 500 mI_ reaction containing 1 mM histidine, 6 mM methyl iodide, 10 pM HMT, 10 mM EgtD was run in 100 mM sodium phosphate buffer at 25 °C for 3 hours. SAH with final concentration of 50 mM was added to initiate the reaction. For control experiment, methyl iodide was added to initiate the reaction. Product formation was monitored by EIPLC. Reaction catalyzed by enzymes preincubated with methyl iodide was 10% slower than that catalyzed by enzymes pre-incubated with buffer.
Figure 14: Conversion of HMTJEgtD cascade under different condition
Conversion of HMT EgtD measured by HPLC (Table 1 , entry 4-9). Area of both substrate (4.6 min) and product (8.6 min) were shown, a) entry 4, b) entry 5, c) entry 6, d) entry 7, e) entry 8, f) entry 9 Figure 15; Conversion of HMTJMT cascade
1H NMR of HMTJMT cascade reaction. Conversion was calculated to be 43% from integrals of reaction mixture by following: C = 2/(2 + 2 )
Figure 16: Conversion of HMT_PMT cascade
1H NMR of HMT_PMT cascade reaction. Spectrum of reaction mixture was shown in a). Signals of product and substrate (b) are overlapping. Conversion was calculated to be 87% from integrals of reaction mixture by following: C = 37 [(1 + 1 ' + 2 + 2')* 1.5]
Figure 17: Conversion of HMT_SgvM cascade
1H NMR of HMT SgvM cascade reaction. Spectrum of reaction mixture was shown in a). No Signal of substrate (b) was observed in reaction mixture. Therefore, the conversion was estimated to be over 99%
Figure 18: Conversion of HMTJ^ovO cascade
1H NMR of HMT_NovO cascade reaction. Conversion was calculated to be 35% from integrals of reaction mixture by following: C = 87 [(1 + 8)*Q 5 + 8 )]
Figure 19: Conversion of HMT-EgtD cascade accepting Deuterated methyl iodide
Conversion of HMT_EgtD cascade accepting Deuterated methyl iodide measured by HPLC Area of both substrate (4.6 min) and product (8.6 min) were shown.
Figure 20: Measured and calculated HR-MS of L-hercynine.
Figure 21: 1H NMR spectrum of L-hercynine.
Figure 22: Measured and calculated HR-MS of L-hereynine-d9.
Figure 23: 1H NMR spectrum of L-hercynine-d9.
Figure 24: 1H NMR spectrum of D-ononitol.
Figure 25: Measured and calculated HRMS of W,/V'-Dimethyl-1 ,4-butanediamine.
Figure 26 Measured and calculated HRMS of compound shown in Figure. Figure 27: Measured and calculated HR-MS of f?-3-methyl-2-oxovalerate.
Figure 28: 1H NMR spectrum of R-3-methyl-2-oxovalerate,
Figure 29: 1NMR spectrum of 2,7-dihydroxy-1 -methylnaphthalene.
Figure 30; Chemoenzymatic synthesis of SAM.
Figure 31: General scheme of a catalytic alkylation system with regeneration of the alkylated carrier.
Figure 32: Cascade reaction coupling Methionine S-methyltransferase (MS T) and 5- thiohistidine N-methyltransferase OvoC
Figure 33: Linear fiting and catalytic efficiency of MSMTRho. Reaction condition was 50 m phosphate buffer (pH 8.0), 20 mM NaCI, 1 mM of constant substrate and 10 mM
MSMTRho·
Figure 34: Extracted ion chromatogram (EIC) of isotope labeled ovothiol A produced by the cascade methyl transfer.
Figure 35. Representative natural products containing b-Me AAs.
Figure 36. natural biosynthetic pathway of b-Me AA. The existing examples includes jS-Me Glu, j3-Me Trp, b-Me Phe, b-Me Leu.
Figure 37. Strategy on stereo-selective /J-methylation of amino acids in this study. Amino acid is converted to a-keto acid by a transaminase (TA), a-keto acid is then stereo- selectively methylated by SAM dependent MT when SAM is regenerate by halide methyltransferase (HMT). Methylated a-keto acid is then converted to b-methyl amino acid by TA.
Figure 38. Synthesis of (2S, 3R)-3-methyl amino adds by combining different TAs and MTs. Structures of products are shown. Figure 39. Synthesis of (2S, 3S)-3-methyl amino acids incorporate isomerase ( arH).
Figure 40. Synthesis of { 25 , 3S)-3-methyl amino acids.
Figure 41. Monofluoromethyl transfer catalyzed by HMT and degradation of F-SAM
Figure 42. Left. HPLC chromatogram of reaction catalyzed by HWTth, Right. HPLC chromatogram of reaction catalyzed by HMTxe.
Figure 43. 1H NMR of monofluoromethyl transfer reaction catalyzed by HMTxe, Resonances of homoserine was assigned with numbers.
Figure 44. HMT-EgtD cascade of monofluoromethyl transfer, a, reaction scheme, b, HPLC monitoring DMH, SAH and F-TMH in the cascade reaction containing 1 mM histidine, 6 mM methyl iodide, 50 mM SAH, 50 mM EgtD and 50 mM HMT. c, Time dependent of product formation of HMT-EgtD cascade.
Figure 45. 1 H NMR of F-TMH formed by HMT-EgtD cascade.
Figure 48. A) Conversion of alpha-keto acids or alpha-hydroxy acids to beta-methyl-alpha hydroxy acids. Involved enzymes: Alcohol dehydrogenase, C-methylating enzyme, HMT. B) Conversion of alcohols to beta-methyl alcohols (or 2-methyl alcohols, for example 2- methylbutyl alcohol). Involved enzymes: Alcohol dehydrogenase, a-keto-acid decarboxylase, C-methylating enzyme, HMT. C) Conversion of amines to beta-methyl amines (or 2-methyl amine, for example 2-methylbutylamine). Involved enzymes: Transaminase, a-keto-acid decarboxylase, C-methylating enzyme, HMT.
Figure 47: S-adenosy!-L-methionine regeneration by the enzyme S-methyl methionine methyl transferase (MSMT) using Se-Methyl-Selenomethionine as methyl donor. MSMT was shown to accept Se-methyl selenomethionine (SeMSeM) as methyldonor to produce SAM from SAH. The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages.
Example 1: S-adenpsyl homocysteine as a methyl transfer catalyst in biocatalvtic methylation reactions
Concept. The following reasoning led us to construct this system, Methyltransferases can transfer a methyl cation from SAM to C-. N-, 0-, P-, S-nucieophiles. The sulfontum moiety of SAM is kineticatly stable so that alkyl transfers only occur in the confinement of enzyme active sites. The thermodynamic reactivity of SAM is high enough to make methylation of almost any nucleophile (N, C, O, P, S) favorable (AG < 0). The high methyl transfer potential of the sulfonium group of SAM is related to the high acidity of S-protonated thioethers (pKa = - 5,4). Most nucleophiles that are subject to enzyme-catalyzed methylation form much weaker acids, making methyl transfers from SAM essentially irreversible. In this regard, halide methyl transferases (HMTs) present a notable exception. In fungi, bacteria, marine algae, diatoms, and halophytic plants HMTs are responsible for the production of methyl halides using SAM as the methyl donor and iodide, bromide and chloride as acceptors (i.e. the native reaction), Hydrogen halides are strong acids (pKa,Hi - 10; pKa,HBr - 8.8; pKa,Hci - 6,4) indicating that methyl transfers from SAM to halides are endothermic. In the biological context, methyl halide production is most likely driven by product removal; the methyl halides evaporate and S-adenosyl homocysteine is degraded by hydrolysis. However, we surmised that under proper in vitro conditions HMT should be able to transfer methyl groups from methyl iodide to SAH {i.e. catalyze the reverse reaction) and therefore provide a simple mechanism for SAM regeneration. In the following we describe the demonstration of this concept.
Halide MTs. In a first step we produced methyl halide transferase from the acidobacterium Chloraddobacterium thernnophilum (HMT) and examined whether this enzyme can convert SAH to SAM under physiological conditions. Protocols for production, purification and analysis of this protein are described in the supporting information (Figure 4). These results confirm that HMT can readily produce SAM from SAH and methyl iodide under physiological conditions (Figures 5 and 6). In a 100 mM phosphate buffer at pH 8 and in the presence of 1 mM methyl iodide, SAH methylation by HMT is characterized by a kca, of 0.05 s”1 and a K , SAH of 0.17 uM (Figure 7). HMT/EgtD cascade. In a second step HMT was combined with EgtD, a histidine methyltransferase from Mycobacterium smegmatis. This SAM-dependent methyltransferase catalyzes trimethylation of the ct-amino group of histidine following a cooperative mechanism. The resulting Na-tri methyl histidine (TMH) is a precursor for ergothioneine biosynthesis in bacteria and fungi. We assembled a reaction to test whether continuous methylation of SAH by HMT could sustain EgtD-catalyzed TMH production (A, Figure 2). Time-dependent product formation was monitored by HPLC (B, Figure 1 ), In a reaction containing 1 mM histidine, 6 mM methyl iodide, 50 mM SAH, and 10 mM of EgtD and HMTthermo in a 100 mM phosphate buffer at pH 8, we observed TMH formation at an initial rate of 0.68 mM/min (C, Figure 2). After two hours this rate decreased significantly, and after 23 hours we determined that about 15 % of histidine was converted to TMH (Table 1 , entry 1 ). This conversion indicated that every SAH molecule was remethylated by HMT nine times. Importantly, a reaction tacking either of the two enzymes produced no detectable TMH {B, Figure 2). This experiment provides the first demonstration that SAH can serve as a catalytic methyl carrier in enzyme catalyzed reactions.
SAH/SAM instability: Why does TMH production come to a halt after 15 % substrate conversion? Closer inspection of the HPLC traces showed that SAH was rapidly hydrolyzed to adenine and ribosyl homocysteine (Figure 3, Figure 8). The rate of this reaction is much faster than spontaneous depurination of SAH (k = 105 s”1}, suggesting that SAH degradation maybe due to SAH nucleosidase (EC 3.2.2.9) contaminations in the HMT and EgtD preparations. Indeed, HMT and EgtD contained 0.013% mol units (0.24 pmol/min) SAH nucleosidase activity per mg protein - even though both proteins appeared highly homogeneous based on SDS PAGE analysis (Figure 4).
Further purification of HMT and EgtD by gel-filtration reduced the SAH nucleosidase activity by 2-fold corroborating the suspicion that the SAH instability is due to contamination (Figure 9). In the presence of re-purified enzymes the half-live of SAH/SAM was significantly increased, allowing the cascade to recycle SAH/SAM up to 30-fold and converting 0.51 mM histidine to TMH, corresponding to a conversion of 51 % (Figure 2E, Table 1 , entry 2). Nevertheless, SAH hydrolysis still limited turnover and yield.
To eliminate this problem completely we produced HMT and EgtD in an E coli strain that is SAH nucleosidase-deficient (KEIO collection, strain JWQ155-1 ). To make this strain compatible with gene expression from a pET28a vector, we deleted the genomic kanamycin- resistant cassette, and introduced a T7 RNA polymerase gene by lysogenesis (Supplementary information). The resulting strain produced HMT and EgtD with a similar efficiency as BL21(DE3) cells. HMT purified from this strain contained no measurable SAH nucleosidase activity (Figure 10), The methylation cascade assembled from these proteins showed less than 5 % SAH degradation after 10 h at 25°C corresponding to a depurination rate of /¾AHjiydroiysis = 2 x 10'5 s 1, consistent with the published rate for the uncatalyzed reaction (Figure 1 1). Instead, the reaction produced TMH at an initial rate of 2.9 pM/min and converted 94% of substrate, corresponding to 56 SAH regeneration cycles (Table 1 , entry 3). Doubling the substrate concentration aiso doubled the yield and the number of SAH regeneration cycles (Table 1 , entry 4). Increasing the concentration of EgtD by two-fold increased substrate conversion to 99 % (Table 1, entry 5), Interestingly, reduction of the SAH/SAM concentration by 2.5-fold did not significantly reduce productivity, and consequently doubled the number of catalytic cycle per SAH (Table 1 , entry 6). Further increases of substrate concentration increased the SAH turnover number to almost 500. In these reactions the percentage of substrate conversion is reduced presumably because of the decreasing ratio between methyl iodide and histidine (Table 1 , entries 7 - 9),
Kinetic analysis of the HMT/EgtD cascade. The following quantitative considerations provide clues as to why the HMT/EgtD cascade reaction (Table 1 , entry 5) works so efficiently. HMT is characterized by a remarkably high substrate affinity (KM. SAH of 0.17 pM) (Figure 7). The steady state concentrations of SAH and SAM were determined to be 2 and 48 mM (Figure 3). Hence, most available SAH is bound to HMT. This is a crucial feature because many SAM-dependent enzymes are subject to product inhibition by SAH. In vitro metbyltransferase assays usually include SAH nucleosidase activity to avoid product inhibition. In cellular systems the activities of MTs are believed to be modulated by the SAH concentration and the SAM/SAH ratio, SAH concentrations are typically maintained at low- or sub-micromolar concentrations by SAH nucleosidases or SAH hydrolases. The high SAH affinity of HMT combined with the high methyl transfer potential of methyl iodide provide a simple way to establish a similar steady state SAH concentration in vitro as in a living cell.
We also examined whether uncatalyzed methyl transfers to solvent or to other reaction components reduce the productivity. Measured rates of methyl iodide hydrolysis in neutral aqueous solutions at 55°C {khydro,ySis = 3 x 106) or at 85X (khydroiy$i$ -
Figure imgf000078_0001
indicate that this reaction is exceedingly slow at room temperature. Indeed, a solution containing 6 mM methyl iodide and 1 mM SAH in 100 mM phosphate buffer at pH 8 produced only 160 mM methanol, 100 pM methyl phosphate, 185 mM dimethyl phosphate after 48 h of incubation at 20°C (Figure 12e). Non-enzymatic methylation of SAH or histidine was not observed (Figure 12e). H ESI-MS analysis of methyl iodide treated HMT and EgtD (Table 2) showed that both enzymes are methylated up to four times. Table 1. Optimization of conversion and number of SAH/SAM regeneration cycles. Reactions were all run in 100 mM phosphate buffer at pH 8.0 (Figure 14). a. Proteins were produced in BL21 (DE3) cell and purified by Ni(ll)-affinity chromatography b. Proteins were produced in BL21 (DE3) cells and purified by Ni(ll)-affinity chromatography and size exclusion chromatography. Histidine CH3I SAH HMT EgtD Conversion No of
Figure imgf000079_0001
Table 2, Calculated and measured MS (incubation at room temperature with or without methyl iodide for 3 hours) of proteins used in Example 1
Figure imgf000079_0002
However, these modifications reduced catalytic activity by less than 10 % (Figure 13). Based on this analysis we conclude that the HMT/EgtD cascade provides a robust and efficient system for methyl transfer from methyl iodide to histidine. Under physiological conditions formation of unwanted methylation products is minimal and does not interfere with product formation. Finally, product inhibition by SAH is suppressed due to efficient re-methylation to SAM by HMT.
Application of other methyltransferases. The Braunschweig Enzyme Database (BRENDA) lists more than 300 SAM-dependent MTs (EC 2.1.1.-) most of which transfer methyl groups to 0-, N- or C- nucleophiles. HMT-mediated SAM regeneration should in principle be applicable to all of these enzymes and provide a general solution for cell-free applications of methyl transferases to large-scale applications. To provide a few test cases we examined the productivity of the following natural product methyl transferases in the context of the SAM regeneration cascade.
Inositol 4-methyltransferase from Mesembryanthemum crystallinum (IMT, EC 2.1.1.129) converts inositol to ononitol (Table 2, entry 3). O-Methyl inositols are plant osmoprotectants, that also have antidiabetic, anti-cancer or anti-inflammatory effects in humans. Inositol methyltransferases reflect the synthetic process of enzyme-mediated methylation in that they afford regiospecific alkylation of one out of six nearly equivalent hydroxyl groups. Chemical approaches for the same transformation include laborious multistep syntheses,
Putresdne N-methyl transferases (PMT, EC2.1.1.53) catalyze the methylation of the diamine putresdne to form the first intermediate in the biosynthesis of alkaloids, including nicotine and tropanes (Table 3, entry 4). Transformation of primary amines to secondary amines by chemical synthesis is often hampered by the higher nucleophilicity of the secondary amine which results in overalkylation. Hence, secondary methyl amines are usually synthesized through indirect routes. In contrast, PMT avoids over-methylation by way of its carefully tailored active site.
C-Methylating enzymes are notable for their abilities of asymmetric alkylation of the beta- carbon of alpha-keto acids (Table 3, entry 5). This activity is of particular interest, since direct asymmetric alkylation alpha to ketones is stilt a challenging objective for synthetic methods.26 As a test enzyme we examined the methyltransferase SgvM from Streptomyces griseoviridis, which can methylate the C3 position of alpha- ketonorvaline, among other substrates. Another class of C-methylating enzymes is represented by the enzyme NovO, which can methylate the sp2-hybridized carbon on the aromatic core of coumarins (Table 3, entry 6J.
These four enzymes (Table 3) were produced and assayed following the same protocols as described for EgtD. The methylated products emerging from the respective HMT/MT cascades were identified and quantified by NMR and ESI-MS (Figures 15 - 29). The reactions containing PMT or SgvM converted 2 mM of their respective substrates, putrescine or a-ketonorvaline almost completely, using 20 mM of SAH in 100 cycles. The enzymes IMT and NovO converted almost half of the substrate. Two more additions of IMT and NovO to the respective reactions drove substrate conversion to near completion (>90 % conversion, Table 3), suggesting that enzyme stability is the limiting factor in these examples. Finally, as a demonstration that HMT/MT cascades could be used for isotope labeling we produced deuterated TMH using deuterated methyl iodide {Table 3, entry 2} using the same conditions as described for production of TMH (Table 3, entry 1). Again, this process afforded almost quantitative conversion of histidine to product. Table 3 Conversion of cascade methyl transfer combining different type of MTs and HMT. Conversion was determined by HPLC or NMR and Numbers of cycle were calculated based on SAH concentration
Substrate Product Conversion Number of cycle
Figure imgf000081_0001
Figure imgf000082_0001
Conclusions, Enzyme cascades consisting of HMT and O-, N- or C-methylating methyl transferases represent simple and robust methylation platforms. Fueled with the off-the-shelf reagent methyl iodide and catalytic concentrations of SAM or SAH these cascades can exploit the exquisite regio-» chemo-, and stereoselectivity of naturally evolved SAM- dependent methyltransferases for in vitro methylation reactions. Based on the results, it is reasonable to expect that radicai-SAM methyltransferases can also be combined with the HMT-dependent SAM regeneration system. Similarly, it can be expected that this methylation technology could be extended to utilize SAM derivatives as a general approach for enzyme catalyzed alkyl transfers.
Supplementary Information to Experiment 1
General information
The gene encoding enzymes were was codon-optimized and synthesized by General Biosystems, Inc. (Morrisville, NC, USA) after which it was cloned (using Ncol/Xhol sites) into the pET-28a plasmid. Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions. The gels were stained with Coomassie brilliant blue.
Analytical high performance liquid chromatography (HPLC) was performed using a Shimadzu LC-20AT HPLC with a Shimadzu SPD-M20A diode array detector. Phenomenex Luna® 5 pm SCX 100 A, LC Column 100 x 4.6 mm was used for cation exchange. Size exclusion chromatography was performed on AKTA Explorer 100 FPLC System with a HiLoad® 16/600 Superdex® 200 pg column. High-resolution mass spectra were obtained on a Bruker maXis 4G UHR-TOF Mass Spectrometer. 1H NMR spectra were recorded on a a Bruker Avance Neo NMR spectrometer operating at 500 MHz proton frequency and chemical shifts are internally referenced to residual proton signals of solvents. Chemical shifts (d) are reported in parts per million (ppm). Standard abbreviations indicating multiplicity were used as follows: s (singlet), d (doublet), t (triplet), dd (doublet of doublets), and m (multiplet). Coupling constants { /) are reported in Hertz (Hz).
Unless otherwise noted, all chemicals and reagents were purchased from Sigma Aldrich and used as recieved. Antibiotics were purchased from PanReac AppliChem. Ingredients for buffers were purchased from Acres Organics. L-Histidine was purchased Fluka™, Myoinositol was purchased from Apollo Scientific ltd. 2,7-dihydroxynaphthalene was purchased from FluoroChem Co. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories.
Protein expression
E. coli BL21(DE3) cell: pET-28a plasmids containing genes were transformed using standard heat- shock protocols for chemically competent E, coli BL21(DE3) cells. E. coli cells containing the plasmid were collected from LB-AGAR plates with Chloramphenicol (35 pg/ml} & Kanamycin (50 pg/ml) and used to inoculate LB medium with Chloramphenicol (35 pg/ml) & Kanamycin (50 pg/ml) (20 ml). After incubation at 37°C overnight, 15 ml of pre-culture was used to inoculate fresh Autoinduction Medium (1 L) with Chloramphenicol (35 pg/ml) & Kanamycin (50 pg/ml). The cells were grown in 3L shaking flask at 37 °C (170 rpm) for about 4 h until OD600 reached 0.6-1.0 and then the culture was cool to 20 °C and grown for 24 h. Cells were harvested by centrifugation at 10,000 x g for 30 minutes and stored at -20 °C.
MTA/SAH nucleosidase knockout E. coli strains: pET-28a plasmids containing genes were transformed by electroporation. Cells containing the plasmid were collected from LB-AGAR plates with Kanamycin (50 pg/ml) and used to inoculate LB medium. After inoculation, expression was done by following the same protocol as that of E. coli BL21(DE3) cell.
For purification, 10 g of cell pellet were suspended in 40 ml lysis buffer (50 mM phosphate, 300 mM NaCI, 10 mM imidazole, pH 8.0). Cells were disrupted by sonication for 3 x 60 s with Branson sonifier 450 (output control 5, 50% duty cycle). Lysates were centrifuged at 10,000 x g for 1.0 h at 4 °C. The cleared lysate was mixed with 1 ml of Ni" NTA agarose at 4 °C for 20 min and loaded onto a column. The agarose beads were washed with 10 ml sodium phosphate buffer containing 10 mM and 20 mM imidazole. The protein was eluted in a lysis buffer solution containing 250 mM imidazole. Protein containing fractions were collected and dialyzed against dialysis buffer (100 mM phosphate, pH 8) aliquoted and stored at -80 °C.
Construction of MTA/SAH nucleosidase knockout E. coli strains that carry inducible T7 RNA Polymerase
During our S-adenosyl methionine regeneration assay, co-factor was converted to adenine as a side reaction. This reaction was catalyzed by MTA/SAH nucleosidase, which is an impurity from protein purification. We still observed the activity of this side reaction after we purified our proteins by size exclusion chromatography. Therefore, an MTA/SAH nucleosidase knockout E. coli strains carrying inducible T7 RNA Polymerase was constructed.
Plasmid pKD46 was transformed to cell strain from the KEIO collection (CGSC #: 8422, MTA/SAH nucleosidase knockout) by electroporation to delete the kanamycin resistant cassette from chromosome. The cells was inoculated onto plate with ampiciliin and incubated at 30 °C. Single colony was picked and suspended in 50 pi LB-medium and then spreaded onto plate without any antibiotic and incubated at 42 °C overnight. The incubation at 42 °C was repeated for three times. Three single colonies were picked and suspended in 50 pi LB- medium and inoculated onto plate without any antibiotic and plates with kanamycin or ampiciliin respectively to verify removal of plasmid pKD46 and deletion of kanamycin resistant cassette. One clone that only grew on plates without any antibiotic was chosen for ADE3 Lysogenization.
Lysogenization was done by following the protocol of the Novagen ADE3 Lysogenization Kit provided by the producer. Firstly, cells from single colonies were grown in LB medium supplement with 0.2% maltose, 10 mM MgS04 at 37 °C to an ODeoo of 0.5. Secondly, 10 pi cultures were mixed with 5 pi of mixture containing 10® pfu ADE3. 10® pfu Helper Phage, and 10® pfu Selection Phage and incubated at 37 °C for 20 min. Then the mixtures were spreaded onto LB plates and plates were incubated overnight at 37 °C. Colonies were randomly picked from plates and used to prepare cell cultures for further verification, storage and protein production. Cascade reaction assay
General HPLC analysis of HMT_EgtD
0.5 ml reaction containing different concentrations of substrates, co-substrate and enzymes were incubated in 100 mM sodium phosphate buffer at at 25 °C. Concentrations of chemicals and enzymes were shown in Figure 1 and Table 1. 30 pi was added to 15 pi of 1 M phosphoric acid to quench the reaction. Quantitative analysis of substrates, product and co- factor was done based on calibration curve established from authentic commercial compound of L-hisfidtne and S-adenosyl homocysteine.
HMTJEgtD and 1H NMR analysis
2 ml reaction containing 100 mM sodium phosphate buffer, 2 mM L-histidine, 20 pM SAH, 12 mM methyl iodide or deuterated methyl iodide, 10 mM HMT and 20 mM MTs were incubated at 25 °C for 72 hours and lyophilized. Reaction mixture was dissolved in 600 pL D2Q for 1H NMR recording.
Figure imgf000085_0001
analysis
2 ml reaction containing 100 mM sodium phosphate buffer, 2 mM 2-oxovaleric add, 20 pM SAH, 4 M methyl iodide, 10 mM HMT and 20 mM SgvM were incubated at 25 °C for 72 hours and lyophilized. Reaction mixture was dissolved in 600 pL D20 for 1H NMR recording. Conversion was determined based on integral of protons from both substrate and product,
HMT_PMT and 1H NMR analysis
2 ml reaction containing TOO mM sodium phosphate buffer, 2 mM N-methyltra nsferase , 20 mM SAH, 8 mM methyl iodide, 10 mM HMT and 20 mM PMT were incubated at 25°C for 72 hours and lyophilized. Reaction mixture was dissolved in 600 pL D2Q for 1H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
HMTJMT and 1H NMR analysis
1 ml reaction containing 100 mM sodium phosphate buffer, 5 mM substrate, 50 pM SAH, 10 mM methyl iodide 10 mM HMT and 40 mM IMT were incubated at 25 °C for 24 hours. Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Supernatant was centrifuged and lyophilized. Reaction mixture was dissolved in 600 pL D20 for 1H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
For product characterization: Firstly, 1 mi reaction was run as before. After 24 hours, Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Additional HMT and IMT were added to the same final concentration and incubated for 24 hours. Centrifugation and addition of new enzymes were repeated once more. After another 24 hours, the reaction was centrifuged and lyophilized. After iyophi!ization, product was dissolved in 600 mί. D20 for 1H NMR recording.
HMT_NovO cascade and 1H NMR analysis 1 ml reaction containing 100 mM sodium phosphate buffer, 5 mM substrate, 50 mM SAH, 10 mM methyl iodide 10 mM HMT and 40 mM NovO were incubated at 25 °C for 24 hours. Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Supernatant was centrifuged and lyophilized. Reaction mixture was dissolved in 600 pL D20 for 1H NMR recording. Conversion was determined based on integral of protons from both substrate and product.
For product characterization: Firstly, 1 ml reaction was run as before. After 24 hours, Reaction mixture was centrifuged at 14,000 x g for 10 minutes and precipitated protein was removed. Additional HMT and NovO were added to the same final concentration. After another 24 hours, the reaction was centrifuged. The solution was extracted 3x with 25ml dichloromethane. The combined organic solutions were evaporated to dryness. Product was dissolved in 600 pL DMSG-d6 for 1H NMR recording.
Characterization of cascade products
L-hercynfne, HRESIM5 (m/z 220 1057 [M+Na f , cafcd for C9H15N3Na02, 220,1056). 1H NMR (D20, pD 8.4, 500 MHz) <¾ 3.19-3 28 (m, 2H), 3.27 (s, 9H), 3.90 (dd, J =4.3, 11 1 Hz, 1 H), 7.00 (s, 1 H), 7.73 (s, 1 H). The 1H NMR spectrum is consistent with that reported in the literature L-hercynine-d9, HRESIMS (m/z 229.1617 [M+Naf, calcd for C9H6D9N3Na02, 229.1621 ). 1H NMR (D20, pD 8.4, 500 MHz) ¾ 3.22 (dd, J =11.1 , 13.6 Hz, 1 H), 3.26 (dd, J = 4.3, 13.6 Hz, 1 H), 3,89 (dd, J =4.3, 1 1.1 Hz, 1 H), 7.00 (s, 1 H), 7.73 (s, 1 H).
L/,LR- Dimethyl-1 ,4-butanediamine, HRESIMS (m/z 117.1386 (M+Hf, calcd for C6H17N2, 117.1386). 1H NMR (DzO, pD 8.4, 500 MHz) dH 1.80 (m, 4H), 2.71 (s, 6H), 3.06 (m, 4H). The 1H NMR spectrum is consistent with that reported in the literature.
R-3- methy l-2-oxova I erate , HRESIMS (m/z 129.0559 [M-H]", calcd for C6H903, 129.0557) 1H NMR (D20, pD 8.4, 500Hz). dH 0.89 (t, J = 7.5 Hz, 3H), 1.09 (d, J - 7.0 Hz, 3H), 1.43-1.50 (m, 1 H), 1.66-1.74 (m, 1 H), 2.89-2.96 (m, 1 H). The 1H NMR spectrum is consistent with that reported in the literature.
D-ononitol, 1H NMR <D20, pD 8.4, 500Hz). dH 3.35 (m, 1 H), 3.38 (m, 1 H), 3.49 (dd, J = 3.0, 10.0 Hz, 1 H), 3.59 (s, 3H), 3.61 (m, 1 H), 3.63 (m, 1 H), 4.04 (dd, J = 2.8, 3.0 Hz, 1 H). The 'H NMR spectrum is consistent with that reported in the literature.
2,7-dihydroxy-l-methylnaphthalene, 1H NMR (DMSO-d6, 500Hz). ¾ 2.28 (s, 3H), 6.82 (dd, J = 2.3, 8.7 Hz, 1 H), 6.90 (d, J = 8.8 Hz, 1 H), 7,03 (d, J = 2.3 Hz, 1 H), 7.44 (d, J = 8.7 Hz, 1 H), 7.58 (d, J = 8.8 Hz, 1 H), 9.29 (s, 1 H), 9.52 (s, 1 H). The 1H NMR spectrum is consistent with that reported in the literature.
Example 2: S-adenosyl-L-methionine regeneration by the enzyme S-methyl methionine methyl transferase (MSMT)
S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs) are ubiquitous in nature, MTs catalyze the methyl group transfer from SAM to C-, 0-, N-, or S-centered nucleophiles and substrates of MTs include nucleic acids and proteins, as well as a lot of secondary metabolites. Those transfers are normally highly chemo- and regio-selective. In some cases of C-methyltransferase, the transfers are stereo-selective as well. Therefore, MTs has significant potential for application in biotechnology because they can be used for methylation of synthetic compounds and natural products, which are often important for the bioactivities of those compounds.
One limiting factor of application of MTs in biocatalysis is the availability of SAM, even though a !ot of researches have been done in microbial production of SAM. In this project, we are aiming to find a new enzymatic way to regenerate SAM from SAH. Methionine S-methy!transferase (MSMT, EC 2.1.1.12) was firstly chosen for SAM regeneration because both donor and acceptor of methyl group of the reaction are sulfides so that activity of this enzyme is more likely to be tuned in favor of reverse reaction. We produced the MSMT from Rhodobacter sp, JA431, and validated the function in vitro. Kinetic parameters have been measured under different conditions. Jccat was measured to be around 10 min 1. Ku was observed to be 9.9 ± 0.6 mM for methionine and 1.5 ± 0.2 mM for SAM.
After the kinetic parameters were measured, a cascade reaction {Figure 32) was set up to verify the concept. 250 pi of reaction were run under 50 mM of phosphate buffer at pH 8.0. Final concentrations of Chemicals and enzymes were as following: NaCI 20 mM, SAH 100 mM, S-methyl methionine 10 mM, TCEP 2 mM, 5-thioIhistidine 1 mM, MSMT and OvoC 10 mM. In negative control, no MSMT was used. In positive control, 300 uM of SAM were used instead of MSMT and SAH. Thiol of ovothiol A was modified with bromo-coumarin. The production of ovothiol A was monitored by detecting the BMC derivative by reverse phase HPLC.
As shown in Figure 33, OvoC transferred a methyl group from SAM and produced ovothiol A in positive control. Around 10 mM of ovothiol A has been produced by the cascade reaction. However, same signal can be observed in the negative control reaction without MSMT. It is theoretically possible that ovothiol A in the cascade reaction can be converted from purified enzymes bounded SAM. To validate that the methyl group was transferred through cascade reaction, LC-MS coupled with isotope labelled SMM was performed. The reaction conditions were as same as the reaction before. Two reactions containing either SMM or SMM-CD3 were run in parallel for UPLC-MS measurement. Ovothiol A standard was injected as well to get the standard retention time of extracted ion chromatogram (EIC) (Figure 2 of Appendix 2 {Experiment 2)).
In the EIC of normal ovothiol A derivative MS 390+, signals can be observed in the samples of standard compound, cascade reaction with normal SMM and SMM-CD3. In contrast, signals of isotope labeled ovothiol A derivative MS 393+ can only be observed in the cascade reaction of SMM-CD3. Therefore, it can be concluded that the deuterium labeled methyl group was transferred by the couple enzymes of MSMT and OvoC from SMM to ovothiol A. S-adenosyl-L-methionine regeneration by the enzyme S-methyl methionine methyl transferase (MSMT) using Se-Methyi-Selenomethionine as methyl donor
MSMT was shown to accept Se-methyl selenomethionine (SeMSeM) as methyldonor to produce SAM from SAH (Figure 47).
Example 3: Applicability of the HMT/MT technology to other aikyltransferase
Based on the results obtained for the HMT/MT technology and MSMT/MT technology, it is reasonable to conclude that the technology is applicable to a range of methylating enzymes.
Quantitative analysis and modelling of the reaction kinetics of HMT/MT cascades can be used to develop optimization strategies. The applicability to further systems will proof the concept that the HMT/MT technology enables scalable and cost-effective solutions for methylation reactions.
It can also be reasonably be expected that the system, such as the HMT/MT system, is also applicable to reactions that transfer chemical groups other than methyl groups, including but not limited to stable methyl isotopologues, ftuoromethyl, ethyl or propargyl groups.
1) Empirical analysis and computational modelling of the HMT/MT technology Specific HMT/MT reactions can be characterized by empirical quantification of time- dependent enzyme activities, the accumulation of protein modifications, and concentrations of substrates, products, intermediates, SAH and SAM. This data can be used to construct a numeric model describing ail kinetic aspects of HMT/MT cascade reactions. To build this model the KinTek Explorer Chemical Kinetics Software can be used. Based on this model predictions can be made as to how the cascade efficiencies change as a response to improving individual c<
Figure imgf000089_0001
ants, or to changing substrates.
2) Optimization of enzyme production and purification
The above quantitative analysis will identify cascade components and reaction conditions that allow a further optimization. The information can be used for the following:
- Protein purification. Al (proteins in Experiments 1 and 2 have been purified via Ni (II) affinity chromatography. For scaling up the method, the cascade reactions may be operated efficiently using enzymes in crude cell-free lysates. Initial experiments suggest that the HMT/MT cascade is relatively insensitive to impurity. Alternatively, a the HMT-MT fusion protein may be attached to a chitin-binding domain (CBD). This tag is widely used for protein immobilization. Chitin-immobilized HMT-MT -CBD could be directly used (and re-used) for methylation reactions. Chitin is a very cost-effective supporting material. Using immobilized enzymes also simplifies the downstream process, because the aqueous solution containing methylated product, volatile buffer salts, sodium iodide and catalytic amounts of SAH/SAM (1 %) can be filtered of and lyophifized to obtain highly pure product as the acetate salt.
- Alternative alkylating agents. Methyl iodide has proven an excellent methyl donor for enzyme catalysed methylation. For large-scale applications the use methyl chloride may be favourable.
Example 4: Direct stereo-selective b-methylation of amino acids by enzymatic cascades linking two co-factor regeneration systems
Furthermore, we demonstrate that the in vitro methylation technology cart be applied for b- methylation of L- and D-ct-amino acids. Analogous schemes can be used for b -methylation of L- and D-a-hydroxy acids, amines and alcohols. This methodology is relevant because stereoselective introduction of b-methyl groups is difficult for traditional methods of organic synthesis.
Methylation is a minor modification, but has profound effect on bioactivities of different molecules in medicinal chemistry. This effect has been given a name of“The Magic Methyl Effect” by medicinal chemist because introducing a methyl group to a small molecule can result in up to 2000-fold boost in potency. Due to this effect, methylation reaction has received remarkable interest in organic synthesis over the past years. Despite the effort scientists have made, asymmetric methylation of non-activated methylene C-H bond remains unsolved.
/8-methyl amino acids (0-Me AA) is a series of non-canonical amino acids, which have been found in non-ribosomal peptides (NRP) and other natural products (Figure 35). The new stereo center of 0-carbon, together with a-carbon, constrains confirmation of the side chain and affect the properties of the molecules that 0-Me AAs compose. It is not surprising that many nature products containing 0-Me AAs are bioactive and some of them, such as daptomycin, are being used in clinic. Furthermore, this effect has been demonstrated in some examples of synthetic peptides, wherein introducing b-Me AAs can prolong the biological activity and result in higher potency and selectivity. Nevertheless, application of this powerful modification in peptide chemistry has been restricted by availability of those amino acids. Up till now, not too many 0-Me AAs are available. As it is challenging to selectively generate two adjacent stereo-centers at once or create a second stereo-center at non-activated position in the presence of the first one, organic synthesis of b-Me AAs requires multiple protecting and de-protecting steps, hazardous conditions and expensive metal catalyst. In term of biocatalysis, enormous success has been achieved on synthesis of /8-Alkyl tryptophan analogues by directed evolution of tryptophan synthase. However, it is limited to tryptophan analogues and is not applicable to other b-Me AAs. Therefore, a comprehensive and concise synthetic route of jS-Me AAs is highly demanded.
In nature, jS-Me AAs are produced via four different approaches. The most common approach comprises three steps. In the first step, amino acid is converted to cr-keto acid by a transaminase (TA) with a-keto glutarate as amine acceptor. Next, er-keto acid is asymmetrically methylated by a SAM-dependent methyltranferase (MT). In the last step, methylated keto acid is converted to jS-Me AAs by the same transaminase of the first step. The existing products of this approach include b-Me arginine, b-Me phenylalanine, b-Me tryptophan, b-Me glutamate and b-Me leucine. Inspired by the natural biosynthetic pathway, we aim to develop an effective enzymatic cascade for stereo-selective b methylation of natural and unnatural amino acids. Enzymatic cascade would offer great economic benefit by avoiding the purification of unstable intermediate and reduce the usage of organic solvents during the purification process. See also Figure 36.
Here, we construct a direct /3-methylation system of amino acids enabled by enzyme cascade reaction (Scheme 1). It combines three enzymes (TA, MT and HMT) and needs two co-factor, pyridoxal phosphate (PIP) and S-adenosyihomoeysteine (SAH). Starting material of this system are simple a-AAs and methyl iodide. We demonstrate that this strategy can be applied to broad scope of amino acids including both L- and D- amino acids by different combinations of TAs and MTs. This system is highly atom economic and produces iodide ion as the only side product. See also Figure 37. Results
Initially, we interrogated the detailed reaction mechanism of natural b-methylation pathway. In the first step, AA is converted to a-keto acid by a transaminase (TA). Since PLP- dependent transaminases has a ping-pong mechanism, ammonia of AA will be trapped within the active site as Pyridoxamine-S'-phosphate if there is no cr-keto glutarate as ammonia acceptor, Meanwhile, the released cr-keto acid can be irreversibly methylated by SAM dependent MT. Subsequently, Pyridoxamine-5’-phosphate bound TA would take b-Me- s-keto acid as substrate and converted to b-Me AA The cofactor PLP is regenerated. On the other hand, the other crucial cofactor, SAM, can be regenerated by halide methyltransferase (HMT), which was discovered by our group recently. Overall, a-keto glutarate would not be essential for catalysis as an amonia carrier. The driving force of this catalytic process is the irreversible methylation of keto acid.
In the first step, we tried to demonstrate the model by making (2S, 3/?)-3-methyl tryptophan, a precursor of Lavendamycin, Maremycins and Strptonigrin, Firstly, we heterologously overexpressed HMT*e from Burkholderia xenovorans, transaminase (MarG) and methyltransferase (Marl) from Streptomyces sp. B9173 in E. coii mtn (DE3) cell and purified all proteins by Ni"-affinity chromatography. Next, we set up 1 ml scale cascade reaction in 100 mM phosphate buffer (pH 8.0) containing 2 mM of L-tryptophan, 4 mM methyl iodide, 40 mM SAH, 40 mM PLP, 40 pM MarG, 40 pM Marl and 20 pM HMTxe. After 18 hours, L- tryptophan (1) was converted quantitatively and (2S, 3R)-3-methyl tryptophan (1a) was the dominant product (Figure 38, Table, entry 1 ), Diastereomer ratio between 3R and 3S isomers was determined to be 93:7 by 1H NMR. The diastereoisomer was produced because of non- enzymatic racemization of b-Me indolpyruvate and both of the enantiomers are substrates of MarG. In principle, it is shown that our design is practical and PLP is tolerant of methyl iodide under this condition.
Next Step, we tried another combination of TA and MT. In this case, branched chain amino acids transaminase (iLvE) is from E.coli and methyltransferase (SgvM) is from Streptomyces griseoviridis. We used L-leucine as first substrate and run the reaction under same condition as we described before. L-leucine was converted to (2S, 3R)-3-methyl leucine quantitatively after 18 hours and Ratio between 3R and 3S isomers was determined to be 96:4 (Figure 38, Table, entry 2).
It was reported that both iLvE and SgvM are promiscuous. Therefore, we tested the iLvE- SgvM system with other AAs. When we used L-norvaline (3) as substrate, product was L- atlo-iso!eucine (3a) and the conversion of 3 to 3a was 50 % {Figure 38, Table, entry 3). When L-norleucine (4) was used as substrate, the conversion to 4a was 82 % (Figure 38, Table, entry 4). Another side chain branched substrate L-2-amino-5-methyl hexanoic acid (5) was converted with conversion of 90%, which is as good as that of native substrate. Surprisingly, both enzymes accept substrates with alkene side chain. For instance, L-allylglycine (§) was methylated with 80% of conversion. When alkene was substituted with two methyl group at the end, L-2-amino-5-methyl-Hex-4-enoic acid (8) was methylated with conversion of 70 %. Compound 8a is a residue of Cyciomarins peptide. Surprisingly, L-methionine (9) was substrate as well and conversion was moderate of 55%. In all the cases, diastereomer ratio was not determined because WMR signals were overlapping.
On the other hand, there is an isomerase (MarH) of methyl indolpyruvate involving in the biosynthesis of (2S, 3S )-3-methyl tryptophan. Therefore, we introduced MarH to our system and final concentration of MarH in the reaction is 40 mM (Figure 39). In this case, L- tryptophan was converted quantitatively as well, but (25, 3S)-3-methyl tryptophan (1b) was the dominant product instead of 1a. Diastereomer ratio between 3S and 3R product was determined to be 91 :9. Figure 39 and 40.
In summary, we have developed a comprehensive enzyme cascade to transfer methyl group directly and stereo-selectively to non-activated b position of amino acids using methyl iodide as methyl donor. This strategy is demonstrated to be applicable to broad scope of amino acids by taking advantage of different combinations of TAs and MTs. The cascade is highly stereo-selective and of great atom economy. The only side product of this cascade is Iodide, which will facilitate the following purification steps. This progress reveals another potential application of SAM regeneration system. More importantly, synthesis of /3-methyl amino acids would enable further application of those compounds in medicinal chemistry and chemical biology studies.
Methods
General methytation condition. 1 ml scale reaction was incubated at room temperature. Reaction was run in 100 mM sodium phosphate buffer (pH = 8.0). Each reaction contains 2 mM of substrate, 4 mM methyl iodide, 40 mM SAH, 40 mM PLP, 40 pM TA, 40 pM MT and 20 pM HMT. After 18 hours, reaction was quenched by addition of 500 pL of chloroform. Chloroform was separated by centrifugation (20000*g, 2 minutes) after vortex. Aqueous layer was collected and iyophilized. Reaction sample was dissolved in 600 pL D2Q after lyophilization. 1H NMR was recorded. Example 5: Enzymatic formation of monofiuoro-SAM and biocatalvtic monofluoromethyl transfer
We further demonstrate that the in vitro methylation technology can be used to produce S- adenosyl fluor-methyl methionine from S-adenosyl homocysteine and fluor-methyl iodide. We also show that this reagent can serve as fluor-methyl donor in a enzyme catalyzed fluor- methylation of a tertiary amine. This result is significant because this is the first demonstration of enzyme catalyzed fluor-methylation.
Over the last decades, fluorine has been well acknowledged in pharmaceutical industry and incorporation of a fluorine atom or fluoroalkyl group into drug molecules has become a routine strategy in drug discovery and development. Encouraged by this demand, organic chemist have developed enormous numbers of techniques for preparation of fluorinated compounds. Comparing to trifluoromethyl (CF3) or difluoromethyl {CF2H) group, methods of direct monofluoromethy!ation (CH2F) are less established in organic synthesis.
To date, there is no enzymatic monofluoromethyl transfer reported. Herein, we report the first example of enzymatic monofluoromethyl transfer including formation of key intermediate of monofluoro-SAM and its application in biocatalysis.
Results.
To test if HMT can transfer monofluoromethyl group, we heterologously overexpressed HMTxe from Burkholderia xenovorans and HMTth from Ch!oracidobacterium thermophilum. in E coli Amtn (DE3) cell and purified both proteins by Ni' -affinity chromatography. Next, we set up HPLC assay monitoring SAH, SAM and F-SAM. Reaction was run in 100 mM sodium phosphate (pH = 8.0) and contains 20 mM CH2FI, 200 pM SAH and 20 pM HMT. Both HMTs were able to transfer monofluoromethyl group (Figure 41 ). Nonetheless, HPLC assay showed that F-SAM was not stable and did not accumulate during the reaction. It degraded and two new peaks were observed by HPLC (Figure 42). To identify the degradation products, we ran a 2 ml scale reaction containing 500 pM SAH, 10 mM CH2FI and 50 pM HMTxe, After the solvent was removed by lyophilization, 1H NMR was recorded. Homoserine was identified based on 1H NMR and the signals were assigned (Figure 43). Perhaps, strong inductive effect of monofluoromethyl group makes the sulfonium unstable and more vulnerable to hydrolysis, which produces homoserine as product. On the other hand, no clear product of the base part was observed. Further experiment needs to be done to identify the product.
Even though F-SAM is not stable, it is clear that F-SAM was formed. To test if F-SAM is stable enough to be accepted by other other methyltransferases, we set up a cascade combing HMTxe and EgtD (Figure 44a). Reaction containing 1 mM histidine, 6 mM methyl iodide, 50 mM SAH, 50 mM EgtD and 50 mM HMT was run in 100 mM sodium phosphate (pH = 8.0). Time-dependent product formation was monitored by high-performance liquid chromatography (HPLC) (Figure 44b). monofluoromethyldimethyl histidine (F-TMH) was observed in the enzyme cascade, while no significant peak was observed in control reaction without HMT*e. After 24h, 98.8 % of DMH was converted to F-TMH (Figure 44c).
To validate the product, a 2 ml scale reaction was run under same condition of analytical assay. After the solvent was removed by lyophilization, the sample was dissolved in 600 mΐ D20 1H NMR was recorded (Figure 45).
F-TMH. 1H NMR (500 MHz, D20, pD 8): <¾ 3.17-3 21 (m, 2H), 3.23 (s, 6H), 3.98 (dd, J = 4.5, 10.7 Hz, 1H), 5.43 (dd, 3 = 6.0, 44.9 Hz, 1 H), 5.49 (dd, J = 6.0, 45.0 Hz, 1 H), 6.92 (s, 1 H), 7.63 (s, 1 H).
In summary, we have developed the first enzymatic monofluoromethy! transfer reaction to produce monofluoro-SAM and demonstrated that the unstable F-SAM can be captured and used as a monofluormethyl donor by other MT, such as EgtD.
REFERENCES
All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

1. A method for alkylating a substrate
which comprises the following steps:
a) an alkylation step wherein a S-alkyltransferase transfers an optionally substituted alkyl group from an alkyl donor to a sulfur- or selenium-containing carrier compound, yielding an alkylated sulfur- or selenium-containing carrier compound;
b) an alkylation step wherein a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur- or selenium-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur- or selenium-containing carrier compound,
and wherein at least a part of the dealkylated sulfur- or selenium-containing carrier compound yielded in step b) is recycled to step a) to regenerate the alkylated sulfur- or selenium-containing carrier compound,
2. The method according to claim 1 , wherein step a) is carried out as a first alkylation step.
3. The method according to claim 1 or 2, wherein each sulfur- or selenium containing carrier compound molecule is subjected to the alkylation step a) on the average at least 2 times, more preferably at least 5 times.
4. The method according to any of claim 1 to 3, wherein, in step a), a S-alkyltransferase transfers the optionally substituted alkyl group from the alkyl donor to a sulfur- containing carrier compound, yielding an alkylated sulfur-containing carrier compound; and
in alkylation step b), a N-, C-, G-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated sulfur-containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated sulfur-containing carrier compound,
and wherein at least a part of the dealkylated sulfur-containing carrier compound yielded in step b) is recycled to step a).
5. The method according to claim 4, wherein the sulfur-containing carrier compound prior to alkylation comprises a thioether moiety.
6 The method according to claim 4 or 5, wherein the sulfur-containing carrier compound prior to alkylation is represented by the following formula (la):
Figure imgf000097_0001
wherein,
R1 is selected from a group consisting of a carboxy group, a hydrogen, a triazote, and an ester group;
R2 is selected from a group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy group;
R3 and R4 are independently hydrogen or a hydroxy group, preferably a hydroxy group; and
B1 is an optionally substituted purine or thienoadenine.
7. The method according to any of claims 4 to 6, wherein the sulfur-containing carrier compound prior to alkylation is selected from the group consisting of S-adenosyl homocysteine (SAH), 2-f!uoroadenosyl homocysteine, S-(5'-adenosyl)-3- thiopropylamine, N6substituted S-adenosyl homocysteine, S-adenosyl N~ acetylhomocysteine, S-adenosyl N,N,-dimethylhomocysteine, S-adenosyl homocysteine methylester, S-adenosyl homocysteine ethylester and S-adenosyl homocysteine phenylester.
8. The method according to any of claims 4 to 7, wherein the sulfur-containing carrier compound prior to alkylation is S-adenosyl homocysteine (SAH),
9. The method according to claim 4, wherein the sulfur-containing carrier compound prior to alkylation is represented by the following formula (lb);
Figure imgf000097_0002
wherein R5 is selected from the group consisting of a methyl group which may be substituted, an ethyl group which may be substituted and a propyi group which may be substituted.
10, The method according to claim 4 or 9, wherein the sulfur-containing carrier compound prior to alkylation is selected from the group consisting of methionine, monofluoromethyl homocysteine, diftuoromethyl homocysteine and trifluoromethyl homocysteine,
11 , The method according to any of claims 1 to 3, wherein, in step a), a S-alkyltransferase transfers the optionally substituted aikyl group from the alkyl donor to a selenium- containing carrier compound, yielding an alkylated selenium -containing carrier compound; and
in alkylation step b), a N-, C-, 0-, S-, or P-alkyltransferase transfers the optionally substituted alkyl group from the alkylated selenium -containing carrier compound to the substrate, yielding an alkylated substrate and a dealkylated selenium -containing carrier compound,
and wherein at least a part of the dealkylated selenium -containing carrier compound yielded in step b) is recycled to step a)
12. The method according to claim 11 , wherein the selenium-containing carrier compound prior to alkylation comprises a selenoether moiety,
13. The method according to claim 11 or 12, wherein the selenium-containing carrier compound prior to alkylation is represented by the following formula (lc):
Figure imgf000098_0001
wherein,
R6 is selected from a group consisting of a carboxy group, a hydrogen, a triazole, and an ester;
R7 is selected from a group consisting of a primary amino group, a secondary amino group, a tertiary amino group, an amide group and a hydroxy;
R8 and R9 are independently hydrogen or a hydroxy group, preferably a hydroxy group; and
B2 is an optionally substituted purine or thienoadenine.
14. The method according to any of claims 11 to 13, wherein the selenium-containing carrier compound prior to alkylation is selected from the group consisting of Se- adenosyl selenohomocysteine, thienoadenosyl selenohomocysteine, Se-(2- fluoroadenosyl) selenohomocysteine, Se-(5'-adenosy!)-3-selenopropylamine, N6- substituted Se-adenosyl selenohomocysteine, Se-adenosyl N-acetyl selenohomocysteine, Se-adenosyl N,N, -dimethyl selenohomocysteine, Se-adenosyl selenohomocysteine methylester, Se-adenosyl selenohomocysteine ethylester and Se- adenosyl selenohomocysteine phenylester, preferably Se-adenosyl selenohomocysteine.
15. The method according to any of claims 1 to 14, wherein the alkyltransferase of step b) is a N-, C-, 0-, or P-alkyltransferase, preferably a N-, C-, or O-alkyltransferase.
16. The method according to any of claims 1 to 15, wherein the alkyltransferase used in step a) is an S-adenosyl methionine (SAM) dependent alkyltransferase.
17. The method according to any of claims 1 to 16, wherein the alkyltransferase used in step b) is an S-adenosyl methionine (SAM) dependent alkyltransferase.
18. The method according to any of claims 1 to 8 and 1 1 to 17, wherein the method is carried out in the absence of S-adenosyl homocysteine (SAH) nucleosidase.
19. The method according to any of claims 1 to 18, wherein the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT), thioether methyltransferases and N-methyl proline methyltransferase, N-dimethyl glycine methyltransferase and N-dimethyl beta-alanine N-methyltransferase, wherein the thioether methyltransferase is preferably selected from methionine S- methyltransferase (MSMT).
20. The method according to any of claims 1 to 19, wherein the alkyltransferase used in step a) is selected from the group consisting of halide methyl transferases (HMT) and methionine S-methyltransferase (MSMT), preferably halide methyl transferases (HMT),
21. The method according to any of claims 1 to 18, wherein the aikyitransferase used in step a) is a carboxylmethyltransferase, preferably a indole-3-acetic acid carboxyl methyltransferase, Trigonelline synthase, Gibberellic acid methyltransferase, salicylic acid carboxyl methyltransferase, Jasmonic add carboxyl methyltransferase, or Caffeine synthase.
22. The method according to claim 19 or 20, wherein the halide methyl transferase (HMT) originates from bacteria, plants, fungi or archaea.
23. The method according to claim 22, wherein the halide methyl transferase originates from an acidobacterium, more preferably from chloracidobacterium thermophilum.
24. The method according to claims 19 or 20, wherein the methionine S-methyltransferase (MSMT) originates from bacteria, plants, fungi or archaea.
25. The method according to claim 24, wherein the methionine S-methyltransferase (MSMT) originates from a rhodobacter, more preferably from Rhodobacter sp. JA431
26. The method according to any of claims 1 to 25, wherein the aikyitransferase used in step b) transfers the optionally substituted alkyl group by a SN2-type reaction.
27. The method according to any of claims 1 to 26, wherein the aikyitransferase used in step b) is selected from the group consisting of
the carminomycin 4-O-methyltransferase DnrK, the demethylrebeccamycm-D-glycose O-methyltransferase RebM, the S-adenosyl-L-methionine-dependent O- methyltransferase SynOMT, the O-methyltransferase BcOMT2, the 2,7-dihydrocy-5- methyl-1 -naphthoate 7-Q-methy!transferase NcsBi , the phosphonate O- methyltransferase Dhpl, the demethyldecarbamoylnovobiocin O-methyltransferase NovP, the methyltransferase CalOI , O-methyltransferase Cal06 the mitomycin 6-0- methyltransferase MmCR, the mycinamicin VI 2”-0-methyltransferase M cE, 10- hydroxycamptothecin O-methyltransferase, the Norbelladine 49-O-Methyltransferase, the phenazine-1 -carboxylate N-methyltransferase PhzM, the dTDP-3-amino-3,4,6- trideoxy-alpha-D-glycopyranose N,N-dimethyltransferase DesVI, the NodS N- methyltransferase, the dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose N,N- dimethyltransferase TylM1 , the Indolethylamine N-methyltransferase, Phenylethanolamine N-methyltransferase, beta-alanine N-methyltransferase, dimethylglycine methyltransferase, Pavine N-Methyltransferase, Psilocybin synthase PsiM, Dimethylallyltryptophan N-methyltransferase, Reticuline N-Methyltransferase, Picrinine N-Methyltransferase, Norajmaline N-methyltransferase, (S)-coclaurine-N- methyltransferase, the Tetrahydroprotoberberine cis-N-Methyltransferase, the cyclopropane-fatty-acyl-phospholipid synthase CPPAS, the methylene-fatty-acyl- phospholipid synthase, the uroporphyrinogen-lll C-methyltransferase UMT, the sterol C-methyltransferase sterol 24-C-methyltransferase, the gamma-tocopherol 5-0- methyltransferase.
28. The method according to any of claims 1 to 27, wherein the the alkyltransferase used in step b) is selected from the group consisting of
the histidine-specific methyltransferase EgtD, the inositol 4-methyltransferase (IMT), the putrescine N-methyl transferase (PMT), the methyltransferase SgvM, the 8- demethylnovobiocic acid C8-methyltransferase NovO and the 5-thiohistidine N- methyltransferase OvoC.
29. The method according to any of claims 1 to 25, wherein the alkyltransferase used in step b) transfers the optionally substituted alkyl group by a radical reaction.
30. The method according to any of claims 1 to 25 and 29, wherein the alkyltransferase used in step b) is selected from the group consisting of Tryptophan 2-C- methyltransferase TsrM, P-methyltra nsferase , Valine methyltransferases PoyC, C!oN6, Pyrrole-2-carboxyl methyltransferase, Fosfomycin Biosynthesis Enzyme Fom3, GenK, Gentamicin biosynthetic methyltransferase.
31. The method according to any of claims 1 to 30, wherein the method is carried out in vitro, preferably in a cell free-system or a buffer.
32. The method according to any of claims 1 to 31 , wherein the method is carried out in vivo.
33. The method according to any of claims 1 to 32, wherein the sulfur- or selenium- containing carrier compound is present in an amount which is substoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate.
34 The method according to any of claims 1 to 33, wherein the alkyl donor is present in an amount which is at least stoichiometric to the amount of the optionally substituted alkyl group to be transferred to the substrate,
35. The method according to any of claims 1 to 34, wherein the alkyl donor is a compound comprising the optionally substituted alkyi group and a leaving group.
38. The method according to any of claims 1 to 35, wherein the alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group, preferably a methyl group.
37. The method according to any of claims 1 to 38, wherein the optional substituent is selected from the group consisting of an alkenyl group, an a!kinyl group, and a fluorine atom.
38. The method according to any of claims 1 to 37, wherein the optionally substituted alkyl group is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyl group, a difluoromethyl group, a trifluoromethyl group, 2-fluoroethyl group, 2,2-dif(uoroethyl group and 2,2,2- trifluoroethyl group, preferably a methyl group and a monofluoromethyl group.
39. The method according to any of claims 1 to 38, wherein the optionaHy substituted alkyl group is isotopically labeled.
40. The method according to any of claims 35 to 39, wherein the leaving group is selected from the group consisting of iodide, bromide, chloride, methionine, selenomethionine, sulfate, dimethylsulfoxide and a cyclic or linear thiaether, preferably iodide, bromide and chloride, more preferably iodide and chloride, even more preferably iodide,
41. The method according to any of claims 1 to 40, wherein the alkyl donor is selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, monofluoromethyl iodide, ethyl iodide, ethyl bromide, ethyl chloride, 1 -monofluoro-2- iodoethane, 1 , 1 -difluoro-2-iodoethane, 1 ,1 ,1 -trifluoro-2-iodoethane , propyl iodide, propyl bromide, propyl chloride, propargyl iodide, propargyl bromide, propargyl chloride, S-methyl methionine, Se-methyl selenomethionine, methyl sulfate, 5-methyl tetrahydrothiophene, S-methyl thietane, S-methyl ethylene sulphide, S-methyl di(monofluoromethyl) sulphide, S-methyl dimethyl sulphide, 2-carboxy tetrahydrothiophene, S-methyl (methylthio)acetic acid, S-methyl 3-(methylthio)propionic acid, trimethylselenonium and trimethylsulfoxonium iodide,
preferably selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, monofluoromethyt iodide, ethyl iodide, ethyl bromide, ethyl chloride, propyl iodide, propyl bromide, propyl chloride, propargyl iodide, S-methyl methionine and Se- methyl selenomethionine.
42. The method according to any of claims 1 to 41 , wherein the alkyl donor is selected from the group consisting of methyl iodide, methyl bromide, methyl chloride, monofluoromethyl iodide and S-methyl methionine, preferably methyl iodide, methyl bromide, methyl chloride, and monofluoromethyl iodide, more preferably methyl iodide, methyl chloride, Se-methyl selenomethionine and monofluoromethyl iodide, even more preferably methyl iodide, Se-methyl selenomethionine and monofluoromethyt iodide.
43. The method according to any of claims 1 to 34, wherein the alkyl donor is represented by the following formula (II):
Figure imgf000103_0001
wherein
R10 is selected from the group consisting of a methyl group, an ethyl group, a propyl group, a propargyl group, a butyl group, a monofluoromethyl group, a difluoromethyl group and a trifluoromethyl group, preferably a methyl group;
X is selected from the group consisting of NH, O and S; and
R10 is selected from an alkyl group or an aryl group, preferably a methyl group or ethyl group.
44. The method according to any of claims 1 to 43, wherein the substrate comprises a nucleophilic atom selected from the group consisting of a nitrogen atom, a carbon atom, an oxygen atom, a sulfur atom and a phosphorus atom.
45. The method according to any of claims 1 to 44, wherein the substrate comprises
a C-H acidic carbon-hydrogen bond; and/or
a group selected from the group consisting of a primary amino group, a secondary amino groups, a tertiary amino group, a hydroxy group, an amide group, a carboxy group, a phosphate group, a phosphonate group, a urea group, thioamide group, thiourea group, thiol group, thione group, disulfide group, phosphine group, a carbonyl group and combinations thereof, preferably primary amino group, a secondary amino groups, a hydroxy group, an amide group, a carboxy group, a carbonyl group and combinations thereof.
46. The method according to any of claims 1 to 45, wherein the substrate is selected from the group consisting of a small molecule with a molecular mass of less than 1 kDa, primary metabolites, secondary metabolites, peptides such as ribosomal peptides, proteins, polysaccharides and nucleic acids,
wherein the primary metabolite is preferably selected from the group consisting of amino acids, nucleosides, nucleotides, lipids, carbohydrates and/or
the secondary metabolites are preferably selected from the group consisting of non ribosomal peptides, polyketides, terpenes, alkaloids, phenylpropanoids, purine and pyrimidine based metabolites.
47. The method according to any of claims 29 to 46, wherein the alkyltransferase used in step b) is a SAM-dependent alkyltransferase, preferably a SAM-dependent methyltransferase, and wherein the sulfur-containing carrier compound is S-adenosyl homocysteine (SAH), and wherein step b) additional yields methionine.
48. The method according to claim 47, wherein at least a part of the methionine yielded in step b) is regenerated by an adenosyl-chloride synthase, which synthesizes S- adenosyl-L-methionine (SAM) from methionine and a co-substrate.
49. The method according to claim 48, wherein the co-substrate is 5-chioro-5-deoxy adenosine.
50. The method according to any of claims 1 to 49, wherein the alkylated substrate yielded in step b) is a b-methylated substrate , which is further converted to a b-methylated product by at least one additional step:
a conversion step c) wherein a converting enzyme converts the b-methy!ated substrate to a b-methylated product.
51. The method according to claim 50, wherein the b-methylated product is selected from the group consisting of a b-methyl-L-a-amino acid, a b-methyl-D-a-a m ino acid, a b- methyl-L-a-hydroxy acid, a b-methyi-D-a-hydroxy acid, a 2-methyl-amine and a 2- methyl-alcohol.
52. The method according to any of claims 50 and 51 ,
wherein the b-methylated substrate is a b-methyl-a-keto acid and the b-methylated product is selected from the group consisting of a b-methyl-L-a-amino acid and a b- methyl-D-a-amino acid, and
wherein conversion step c) is a transamination wherein the converting enzyme is a transaminase that converts the b-methyl-a-keto acid to a b-methyl-amino acid as the b- methylated product.
53. The method according to claim 52, comprising a further conversion step d ) after step b) and before step c), wherein step d ) is an isomarization step wherein an isomerase converts one isomere of the b-methylated a-keto acid to a different isomere.
54. The method according to any of claims 1 to 53, comprising at least one additional step: a conversion step d) wherein at least one converting enzyme converts a presubstrate to the substrate.
55. A kit for alkylating a substrate comprising a S-alkyitransferase as defined in any of claims 1 , 16 and 19 to 25, a N-, C-, 0-, S-, or P-alkyltransferase as defined in any of claims 1 , 15, 17, 26 to 30 and 47 to 49, an alkyl donor as defined in any of claims 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of claims 1 and 3 to 14.
56. The kit according to claim 55, further comprising a substrate as defined in any of claims 44 to 46.
57. The kit according to claim 55, further comprising a presubstrate and a converting enzyme, which converts the presubstrate to the substrate.
58. The kit according to claim 57, wherein the presubstrate is selected from the group constisting of amino acids, a-hydroxy acids, a-keto acids, amines, alcohols and ketones, preferably from the group consisting of amino acids, a-hydroxy acids and ketones.
59. The kit according to claim 57 or 58, wherein the converting enzyme is selected from the group consisting of a transaminase, alcohol dehydrogenase and a-keto acid decarboxylase,
60. The kit according to any of claims 55 to 59, which is substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2,2.9).
61. The kit according to any of claims 55 to 60, further comprising an adenosyl-chloride synthase and a co-substrate, which is preferably 5-chloro-5-deoxy adenosine.
62. Use of a S-alkyltransferase as defined in any of claims 1 , 16 and 19 to 25, a N-, C-, 0-, S-, or P-alkyltransferase as defined in any of claims 1 , 15, 17, 26 to 30 and 47 to 49, an alkyl donor as defined in any of claims 1 and 35 to 43 and a sulfur- or selenium- containing carrier compound as defined in any of claims 1 and 3 to 14 or a kit as defined in any of claims 55 to 61 for the production of an alkylated substrate
63. The use according to claim 62 for the production of an alkylated substrate, wherein the substrate prior to alkylation or the alkylated substrate is as defined in any of claims 44 to 46.
64. The use according to claim 62 or 63 for the production of an alkylated substrate, wherein the alkylated substrate is isotopically labeled.
65. The use according to any of claims 62 to 64 for the production of an alkylated substrate in vitro, preferably in a cell-free system or a buffer.
66. The use according to any of claims 62 to 65 for the production of an alkylated substrate in vivo,
67. The use according to any of claims 62 to 66 for the production of an alkylated substrate in a bacterial cell, plant cell or fungal cell,
68. The use according to any of claims 66 to 67for the production of an alkylated substrate in a bacterial cell, wherein the bacterial cell is preferably E.coii or C. glutamicum.
69. The use according to any of claims 66 to 68 for the production of an alkylated substrate, wherein the alkylated substrate is isolated from a cell.
70. A method for producing an alkylated sulfur- or selenium-containing carrier compound, comprising an alkylation step, wherein a S-aikyltransferase as defined in any of claims 1 , 16 and 19 to 25 transfers an optionally substituted alkyl group as defined in any of claims 1 , 36 to 39 from an alkyl donor as defined in any of claims 1 and 35 to 43 to a sulfur- or selenium-containing carrier compound as defined in any of claims 1 and 3 to 14, yielding an alkylated sulfur- or selenium-containing carrier compound.
71. The method according to claims 70, wherein the method is carried out in vitro, preferably in a cell-free system or a buffer.
72. The method according to claims 70 or 71 , wherein the alkylated sulfur- or selenium- containing carrier compound is isolated and optionally purified.
73. The method according to any of claims 70 to 72, wherein the optionally substituted alkyl group is a methyl group or a monofluoromethyl group.
74. The method according to any of claims 70 to 73, wherein the sulfur- or selenium- containing carrier compound is a sulfur-containing carrier compound, preferably 8- adenosyl homocysteine (SAH) and the alkylated sulfur- or selenium-containing carrier compound is a methylated sulfur-containing carrier compound, preferably S-adenosyl- methionine (SAM).
75. The method according to any of claims 70 to 74, wherein the method is carried out in the absence of S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2 9).
76. The method according to any of claims 70 to 75, wherein the S-alkyitransferase is a halide methyl transferase, more preferably a halide methyl transferase from an acidobacterium, even more preferably a halide methyl transferase from chloracidobacterium thermophilum.
77. The method according to any of claims 70 to 76, wherein the alkyl donor is methyl iodide, methyl bromide, methyl chloride, Se-methyl selenomethionine or monofluoromethyl iodide, preferably methyl iodide, methyl chloride, Se-methyl selenomethionine or monofluoromethyl iodide, more preferably methyl iodide or monofluoromethyl iodide.
78. The method according to any of claims 70 to 75, wherein the S-alkyltransferase is a methionine S-methyltransferase (MSMT), preferably a methionine S-methyltransferase from a rhodobacter, more preferably from Rhodobacter sp. JA431.
79. The method according to any of claims 70 to 75 and 78, wherein the alkyl donor is S- methyl methionine.
80. A kit for producing an alkylated sulfur- or selenium-containing carrier compound comprising a S-alkyttransferase as defined in any of claims 1 , 16 and 19 to 25, an alkyl donor as defined in any of claims 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of claims 1 and 3 to 14.
81. The kit according to claim 80 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the sulfur- or selenium-containing carrier compound and the alkylated sulfur-containing carrier compound are as defined in claim 66.
82. The kit according to claim 80 or 81 , which is substantially free from S-adenosyl homocysteine (SAH) nucleosidase (EC 3.2.2.9).
83. The kit according to any of claims 80 to 82 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the S-alkyltransferase is as defined in claim 76.
84. The kit according to any claims 80 to 83 for producing an alkylated sulfur- or selenium- containing carrier compound, wherein the aikyl donor is as defined in claim 77.
85. The kit according to any of claims 80 to 82 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the S-alkyltransferase is as defined in claim 78.
86. The kit according to any claims 80 to 82 and 85 for producing an alkylated sulfur- or selenium-containing carrier compound, wherein the alkyl donor is as defined in claim 79.
87. Use of a S-alkyltransferase as defined in any of claims 1 , 16 and 19 to 25, an alkyl donor as defined in any of claims 1 and 35 to 43 and a sulfur- or selenium-containing carrier compound as defined in any of claims 1 and 3 to 14 or a kit as defined in any of claims 80 to 86 for the production of an alkylated carrier compound.
88. The use according to claim 87, for the production of an alkylated carrier compound in vitro, preferably in a cell-free system or a buffer.
89. The use according to claim 87 or 88, wherein the alkylated sulfur- or selenium- containing carrier compound is isolated and optionally purified.
90. The use according to any of claims 87 to 89, wherein the sulfur- or selenium-containing carrier compound and the alkylated sulfur- or selenium-containing carrier compound are as defined in claim 74.
91. use according to any of items 87 to 90, wherein the S-a!kyltransferase is as defined in claim 76.
92. The use according to any of claims 87 to 91 , wherein the alkyl donor is as defined in claim 77.
93. The use according to any of claims 87 to 90, wherein the S-alkyitransferase is as defined in claim 76.
94. The use according to any of items 87 to 90 and 92, wherein the alkyl donor is as
defined in claim 77.
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