WO2000032623A1 - Catalytic peptides consisting of a designed helix-loop-helix motif and their use - Google Patents
Catalytic peptides consisting of a designed helix-loop-helix motif and their use Download PDFInfo
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- WO2000032623A1 WO2000032623A1 PCT/SE1999/002238 SE9902238W WO0032623A1 WO 2000032623 A1 WO2000032623 A1 WO 2000032623A1 SE 9902238 W SE9902238 W SE 9902238W WO 0032623 A1 WO0032623 A1 WO 0032623A1
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- catalytic
- peptide
- ala
- naturally occurring
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
Definitions
- the present invention relates to new catalytic pep- tides and catalysts for ammo acid synthesis.
- the inventor of the present invention has, together with co-workers, earlier in two articles, J. Chem. Soc. Perkin. Trans. 2, 1998, 10, 2271-2274 and Chem. Commun., 1998, 15, 1547-1548, described folded polypeptide motifs, one for decarboxylation of oxaloacetate and one for use in mimicking enzymatic transammation.
- these folded polypeptides differs in many ways from the catalytic peptides according to the invention.
- Most lmpor- tantly the peptide described the latter publication has only demonstrated an ability to bind the cofactor pyridoxal phosphate m a non-productive mode, i.e.
- the cofactor forms an lmine with a lysine side chain.
- the cofactor In order to catalyse the transammation reaction the cofactor must release the lysine side chain and form such a bond with the ammo group of the ammo acid to be deammated.
- the peptide reported in Chem. Comm. has not demonstrated the key function of the transammation catalysts of the present invention, i.e. the ability to to bind the the intermediate, formed from the ammo acid and the cofactor, by non-covalent forces m a conformation where residues m the sequence of the catalyst, exposed on the surface of the folded peptide, catalyses the chemical transformations.
- the object of the present invention is to provide a new catalytic system specially designed for the catalysis of ammo acid synthesis, particularly synthesis of amino acids not found in nature, said system being more efficient than the ones existing today. More precisely, the invention provides new catalytic peptides and catalysts suitable for catalysis of the transammation and/or de- carboxylation reactions in synthesis of ammo acids. Furthermore, the catalysts according to the invention can be designed to have high specificity.
- the present invention thus relates to a new catalytic peptide consisting of a designed helix-loop-helix motif, and to catalysts comprising two dimerised peptides of this kind.
- One advantage of the present invention is the ability of the catalytic peptides according to the invention to bind key intermediates along the reaction pathway by non-covalent bonds and thus catalysing the transamnation reaction.
- peptide catalysts reported here show enhanced and synergistic activity when used m the forms of heterodimers, i.e. when both the de- carboxylase mimetic and the transammase mimetic are used simultaneously (further illustrated m figure 6 B) .
- Ki- netic evidence shows that synergistic effects are obtained between the two, and that heterodimers are more efficient catalysts.
- the catalytic peptide according to the invention thus consists of a helix-loop-helix motif, consisting of at least 37 ammo acids and have lysine (Lys) , arginine (Arg) , histidine (His) , glutamic acid (Glu) , or aspartic acid (Asp), independently of each other, m positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37.
- Lys and Arg are particularly suitable for production of acid ammo acids, while Glu and Asp m combination with His, Arg or Lys are particularly suitable for production of basic ammo ac- ids. These ammo acids are exposed on the surface of the motif. Ammo acids with uncharged side chains are also produced by these peptides.
- a preferred catalytic peptide according to the invention consists of 42 ammo acids.
- the ammo acids in positions 12 and 31 are leucine (Leu) , in position 9 isoleucine (lie) , in positions 35 and 38 phenylalanine (Phe) , and in positions 5, 16 and 27 norleucine (Nle) .
- Nle can be replaced by Leu without loss of activity.
- a preferred embodiment of the catalytic peptide according to the invention thus has a sequence according to SEQ ID NO: 1 given m the appended sequence listing.
- the N- terminal and/or the C-termmal of the peptide are "capped", which herein means that the ammo group of the ammo acid at the N-termmal is acetylated, which is denoted m the se- quences by Ac, and that the carboxy group of the am o acid at the C-termmal is amidated, which is denoted m the sequences by NH 2 .
- the general sequence of a capped peptide according to the invention is SEQ ID NO: 2 given the appended sequence listing.
- Two especially preferred catalytic peptides according to the invention are the peptides with SEQ ID NO : 3 and SEQ ID NO : 5 given in the appended sequence listing, and the "capped" version of these peptides, i.e. the pep- tides with SEQ ID NO: 4 and SEQ ID NO: 6 given in the appended sequence listing.
- the peptides according to the invention may e.g. be produced with conventional chemical synthesis or by genetic engineering, e.g. m a host such as E. coll.
- the invention also relates to catalytic molecules and proteins comprising the above mentioned sequences.
- Such a catalytic substance may be the result of incorporation of the catalytically active ammo acids, i.e. the histidmes, lysmes, argm es, glutamic acids, and/or aspartic acids in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37, into a larger molecule, preferably a naturally occurring protein, m such a way that the ammo acid in those positions retain the same spatial geometry as they have m the catalytic peptides according to the invention.
- the catalytically active ammo acids i.e. the histidmes, lysmes, argm es, glutamic acids, and/or aspartic acids in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37
- the catalyst according to the invention comprises two catalytic peptides in the form of helix-loop-helix motifs according to the invention which are dimerised to form a four-helix bundle.
- the catalyst according to the invention consists only of such a homodimenc four-helix bundle.
- the whole sequence of the catalytic peptide according to the invention can be fused to a naturally occurring protein of high stability to form a so called "fusion protein", the whole sequence of the catalytic peptide according to the invention can be incorporated into a naturally occurring protein by replacing a helix- loop-helix motif that already exists. It is also possible to incorporate the catalytic residues, i.e.
- the catalyst according to the invention may also be a non-peptidic molecule wherein the functional groups of the side chains of the catalytic residues in said positions are in the same relative positions as in the catalytic peptide according to the invention.
- Such a catalyst will include amino groups, guanidino groups and imidazoyl groups in the same relative positions as the side chains of lysines, arginines and histidines in the helix-loop-helix motif according to SEQ. ID No. 1.
- the catalyst according to the invention may function as a transaminase mimetic, i.e. as a catalyst of a transammation reaction.
- a preferred embodiment of a transami- nation catalyst according to the invention is a dimerised four-helix bundle consisting of two helix-loop-helix motifs, the sequence of each being SEQ. ID NO. 3 or .
- the efficiency of the catalyst can be further enhanced by replacing one of the transaminase mimetic motifs with a second catalytic peptide functioning as a decarboxylase mimetic, i.e. as a catalyst of a decarboxylation reac- tion.
- a preferred embodiment of a catalyst according to the invention functioning both as a transami- nase mimetic and a decarboxylase mimetic is a dimerised four-helix bundle consisting of one motif, the sequence of which is SEQ. ID NO. 3 or 4 , and one motif, the sequence of which is SEQ. ID NO. 5 or 6 , which is further illustrated below.
- the decarboxylase function may be incorporated into the helix-loop-helix motif that catalyses the transammation reaction.
- This catalyst will provide efficient transamination to continuously regenerate the pyridoxamine phosphate needed for the catalytic amination of ⁇ -keto acids to form amino acids .
- the production of many amino acids is based on the use of the pyridoxal phosphate cofactor in the transamination reaction.
- the catalysts according to the in- vention are used, it is possible to use the naturally occurring form of the pyridoxal phosphate cofactor instead of modified, synthetic versions of this cofactor. This feature is an important advantage or the invention because the synthesis of such derivatives for commercial use is often expensive and such cofactors may not be able to find the optimum geometry for transamination with optimum efficiency.
- the binding of the pyridoxal phosphate cofactor and intermediates in pyridoxal phosphate mediated reaction have been shown in the present invention to be controlled by the non-covalent interactions between the phosphate group of the cofactor, other charged substituents of the pyridoxal phosphate derived intermedi- ates and arginine or lysine residues on the surface of the folded motif.
- a key feature of the present invention is thus the ability of the catalytic peptides to bind the aldimine and ketimme intermediates and to catalyse stereospecifically the 1,3 -proton transfer that mtercon- verts these intermediates.
- the transaminase mimetic according to the invention is rich in argmmes and lysmes that bind the m- termediates purely by non-covalent forces.
- the transamination catalyst has histidine and lysine residues.
- Fig. la is a UV-spectrum illustrating a transammation reaction catalysed by a catalyst according to the invention.
- Fig. lb is a X H-NMR spectrum illustrating a transamma- tion and decarboxylation reaction catalysed by a catalyst according to the invention.
- Fig. 2 illustrates the coupling of the transammation and the decarboxylation reactions performed with the catalyst according to the invention.
- Fig. 3 illustrates the reaction mechanism for transamination of aspartic acid to oxaloacetate .
- Fig. 4 illustrates the reaction mechanism for decarboxylation of oxaloacetate.
- FIG. 5 illustrates the reaction rates for transamma- tion of aspartic acid to oxaloacetate, catalysed with a peptide according to the invention.
- Fig. 6 A illustrates the reactivity of the peptide with SEQ ID NO: 4 in transammation.
- Fig. 6 B illustrates the reactivity of the peptide with SEQ ID NO: 4 n combination with the peptide with SEQ ID NO: 6 m transammation.
- Fig. 7 illustrates the enantioselectivity of the peptides according to the invention in transammation.
- Fig. 8 is a modelled structure of the peptide with SEQ ID NO: 4.
- Fig. 9 is a modelled structure of the dimer formed by the peptide with SEQ ID NO: 3 and the peptide with SEQ ID NO: 6.
- a catalyst according to the invention the peptide with SEQ ID NO: 3, was synthesised on a Fmoc-Gly-PEG-Ps polymer (PerSeptive Biosystems) using a PerSeptive Bio- systems Pioneer automated peptide synthesiser and a standard Fmoc chemistry protocol.
- the peptide was cleaved from the polymer and deprotected with TFA (10 ml) , an- isole (220 ⁇ l) , ethanedithiol (333 ⁇ l) and thioanisole
- peptides 4 and 6 were synthe- sised in the same way except that the final step before cleavage from the resin is an acetylation of the free N- terminal amino group by acetic anhydride.
- the choice of polymer linker decides whether the C-terminal is capped or not.
- PAL-PEG-PS was used to obtain C-terminal amides
- PAC-PEG-PS was used to obtain the free acids.
- FIG. 8 A modelled structure of peptide 4 is shown in figure 8.
- the model shows docked aldimine intermediate and Arg, Lys and His residues involved in catalysis. Only one monomer is shown for reasons of clarity, but the folded peptide is a dimer.
- a catalyst consisting of two dimerised peptides 4 was used to catalyse the transamination reaction of as- partic acid to form pyridoxamine phosphate and oxaloacetate.
- the transamination reaction is a multistep reversible reaction and at equilibrium the mixture of species coexist.
- the reaction can however be driven to completion by an oxaloacetate decarboxylase that consumes one of the reaction products, as illustrated below in example 3.
- the reaction between peptide 4, pyridoxal phosphate and aspartic acid was monitored by UV-spectroscopy, and the result is shown in figure la.
- the UV-spectrum shows the pyridoxal phosphate absorbance at 390 nm and the pyridoxamine phosphate/ketimine absorbance at 335 nm.
- the transamination reaction from example 2 was directly coupled to the decarboxylation re- action.
- the complete reaction scheme is illustrated in figure 2, and in more detail in figure 3, showing the reaction mechanism for transamination of aspartic acid to form oxaloacetate, together with figure 4, showing the reaction mechanism for the decarboxylation of oxalo- acetete .
- the oxaloacetate, i.e. the ⁇ -ketoacid of aspartic acid, produced by the transamination reaction described in example 2 is decarboxylated to form pyruvate.
- This re- action can be catalysed by the catalysts according to the invention.
- the catalyst consists of peptide 4, as the transaminase mimetic, and peptide 6, as the decarboxylase mimetic.
- the pyruvate formed in the decarboxylation reaction is then aminated by the pyridoxamine phosphate produced in the transamination reaction and forms alanine. It was found that more than 5 equivalents of alanine was formed per 1 equivalent of peptide.
- the catalyst according to the invention is thus capable of many turnovers. The maximum number of turnovers per catalytic peptide may not be limited to 5, even though this was used in the example.
- the efficiency of peptide 4 in transammation under conditions of a 20-fold excess of ammo acid over peptide catalyst was compared to a reference catalyst, comprising 3 Arg, 1 Lys and 1 His, but having a configuration different from peptide 4.
- the sequence of the reference catalyst was :
- the transammation was a further 3 times faster than with L-Asp and thus more than 3000 times faster that the reaction of L-Asp catalysed by the reference peptide. It is thus clear that the catalyst according to the invention is capable of discrimination between enatiome ⁇ c substrates, which is further illustrated m example 7.
- Example 5 In this example the reactivity of the catalytic peptides according to the invention was studied.
- the peptide according to the invention used was peptide 4, and it was used to catalyse transamination of aspartic acid, leading to formation of oxaloacetate.
- the reaction mechanism for this transamination is shown in figure 3.
- reaction rates were measured by following the disappearance of pyrodixal phosphate at 390 nm and the appearance of pyridoxamine phosphate at 330 nm.
- Peptides similar to the peptides according to the invention were also studied. It was found that the peptides with the following sequences show no measurable production of pyridoxamine phosphate within 20 minutes (1200 s) and are thus not reactive. These peptides contain arginines and histidines but no lysines.
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Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000585264A JP2002531081A (en) | 1998-11-30 | 1999-11-30 | Catalytic peptide composed of designed helix-loop-helix motif and use thereof |
CA002353078A CA2353078A1 (en) | 1998-11-30 | 1999-11-30 | Catalytic peptides consisting of a designed helix-loop-helix motif and their use |
AU20153/00A AU2015300A (en) | 1998-11-30 | 1999-11-30 | Catalytic peptides consisting of a designed helix-loop-helix motif and their use |
EP99963783A EP1144445A1 (en) | 1998-11-30 | 1999-11-30 | Catalytic peptides consisting of a designed helix-loop-helix motif and their use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE9804137A SE9804137D0 (en) | 1998-11-30 | 1998-11-30 | New peptides |
SE9804137-9 | 1998-11-30 |
Publications (1)
Publication Number | Publication Date |
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WO2000032623A1 true WO2000032623A1 (en) | 2000-06-08 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/SE1999/002238 WO2000032623A1 (en) | 1998-11-30 | 1999-11-30 | Catalytic peptides consisting of a designed helix-loop-helix motif and their use |
Country Status (6)
Country | Link |
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EP (1) | EP1144445A1 (en) |
JP (1) | JP2002531081A (en) |
AU (1) | AU2015300A (en) |
CA (1) | CA2353078A1 (en) |
SE (1) | SE9804137D0 (en) |
WO (1) | WO2000032623A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001085906A2 (en) * | 2000-05-05 | 2001-11-15 | Modpro Ab | Catalytically active peptides |
-
1998
- 1998-11-30 SE SE9804137A patent/SE9804137D0/en unknown
-
1999
- 1999-11-30 AU AU20153/00A patent/AU2015300A/en not_active Abandoned
- 1999-11-30 JP JP2000585264A patent/JP2002531081A/en active Pending
- 1999-11-30 EP EP99963783A patent/EP1144445A1/en not_active Withdrawn
- 1999-11-30 CA CA002353078A patent/CA2353078A1/en not_active Abandoned
- 1999-11-30 WO PCT/SE1999/002238 patent/WO2000032623A1/en not_active Application Discontinuation
Non-Patent Citations (2)
Title |
---|
MALIN ALLERT ET AL.: "A designed folded polypeptide model system that catalyses the decarboxylation of oxaloacetate", J. CHEM. SOC., PERKIN TRANS.,, vol. 2, 1998, pages 2271 - 2274, XP002947472 * |
MALIN ALLERT ET AL.: "Non-covalent control of site-selective incorporation of the pyridoxal phosphate cofactor into a folded polypeptide motif-mimicking a key step in enzymatic transamination", CHEM COMMUN.,, vol. 15, August 1998 (1998-08-01), pages 1547 - 1548, XP002947471 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001085906A2 (en) * | 2000-05-05 | 2001-11-15 | Modpro Ab | Catalytically active peptides |
WO2001085756A3 (en) * | 2000-05-05 | 2002-03-14 | A & Science Invest Ab | Site-selective acyl transfer |
WO2001085906A3 (en) * | 2000-05-05 | 2002-10-03 | A & Science Invest Ab | Catalytically active peptides |
US7230072B2 (en) | 2000-05-05 | 2007-06-12 | Modpro Ab | Site-selective acyl transfer |
US7364889B2 (en) | 2000-05-05 | 2008-04-29 | Modpro Ab | Catalytically active peptides |
Also Published As
Publication number | Publication date |
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AU2015300A (en) | 2000-06-19 |
SE9804137D0 (en) | 1998-11-30 |
JP2002531081A (en) | 2002-09-24 |
EP1144445A1 (en) | 2001-10-17 |
CA2353078A1 (en) | 2000-06-08 |
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