WO2007020428A1

WO2007020428A1 - Novel peroxidases and uses

Info

Publication number: WO2007020428A1
Application number: PCT/GB2006/003045
Authority: WO
Inventors: Andrew Trevor Smith; Emile Ngo
Original assignee: University Of Sussex
Priority date: 2005-08-15
Filing date: 2006-08-15
Publication date: 2007-02-22
Also published as: GB0516699D0; GB2429709A

Abstract

Haem-based peroxidases modified at positions (38, 41) and (42), in relation to horseradish peroxidase, show stereoselectivity and activity similar to that of the cytochromes P450 and chloroperoxidase, but without the necessity for ancillary enzymes or reagents.

Description

NOVEL PEROXIDASES AND USES

The present invention relates to novel peroxidases and uses therefor.

The selective transfer of an oxygen atom to a substrate can often be difficult to achieve chemically. Organic synthesis for the purpose of producing high value pharmaceutical products relies on a range of complex strategies to achieve this, sometimes with added synthetic complexities and poor yields. Biologically, specific enzymes in an aqueous environment achieve these kinds of reactions, usually with very high specificity, but often have limited catalytic robustness leading to poorer yields.

One class of enzyme that achieves this kind of chemistry includes the haem-based cytochromes P450, a diverse group of selective monooxygenases. These are complex yet quite sensitive enzymes, often with quite low catalytic turnover, that normally require ancillary reductases in order to activate molecular oxygen in a concerted manner, within a highly structured haem active site.

Truly selective oxygenation reactions can be difficult to achieve synthetically in high yield, yet biologically the cytochrome P450 dependent hydroxylases are capable of catalysing chemically difficult oxygen atom insertions, into compounds that may otherwise be regarded as difficult to activate, chemically. In the catalytic cycle of both peroxidases and P-450's a large body of indirect evidence has suggested that the Fe^IV=O / porphyrin or protein radical may be a common intermediate at a crossroads in their respective mechanisms. Very recently, fresh attempts have been made to trap such intermediates cryogenically and determine their X-ray structures, both for horseradish peroxidase and cytochrome P450cam; in both cases, without bound substrate. While the catalytic competence of the peroxidase intermediates has long been established, the same has not yet been unambiguously demonstrated for the putative ferryl (Fe^IV=O) porphyrin / protein, radical intermediate of cytochrome P450. Moreover, for a related reaction catalysed by haem oxygenase, the crucial intermediate has now been unambiguously shown to be Fe³⁺-O-O-H not an FeIV=O species as previously assumed. Although the cytochrome P450 enzymes offer unique specificity in their catalysis of oxygen atom transfer reactions, they are not robust catalysts and require additional ancillary reductases in order to activate molecular oxygen. Cytochromes P450 can also utilise hydrogen peroxide to replace direct oxygen activation in sterioselective oxidation, however the reaction is slow and results in rapid inactivation of the enzyme and a few turnovers

In contrast, plant peroxidases are simple robust catalysts able to utilise a cheap soluble oxidant, hydrogen peroxide. Unfortunately, they do not normally catalyse oxygenation reactions within the active site without modification of their relatively 'closed' active site architecture. Even then, these reactions are slow, relative to the P450's, and often not very enantioselective.

Thus, there is a need for a robust peroxygenase.

We have now identified mutations that engineer the haem active site cavity of haem- based peroxidases, such as horseradish peroxidase and Coprinus Cinereus peroxidase, robust plant and fungal enzymes respectively, that thereby allow them to effect oxygen atom transfer chemistry analogous to that performed by cytochromes P450 and chloroperoxidase.

Accordingly, in a first aspect, the present invention provides a haem-based peroxygenase enzyme, wherein positions R38, F41 and H42, by comparison with accompanying SEQ ID NO. 1, are substituted, and wherein the arginine at position 38 is substituted by a smaller residue, the phenylalanine at position 41 is substituted by a smaller, neutral residue and the histidine at position 42 is substituted by another polar, or neutral residue.

Preferably, histidine at position 42 is substituted by another polar, residue, preferably an acidic residue.

Surprisingly, substitution of all three residues simultaneously has been found to leave the catalytic cleft in a high spin state, thereby enabling immediate catalytic activity on contact with hydrogen peroxide, or similar peroxide source, and the substrate to be peroxygenated. Preferably, a further substitution located at position 70, where the wildtype asparagine is replaced by histidine (N70H) is also beneficial under certain circumstances when incorporated with the three mutations above.

Position 38 is sterically blocked when occupied by the native arginine, and smaller residues allow better access to the peroxygenation site by substrate. Although histidine may be used at this position, and is encompassed by the present invention, the side chain of histidine is still somewhat bulky, although not as bulky as the arginine side chain, and only has the one point of flexibility. Thus, access to the catalytic cleft is improved by comparison with arginine, but there is still some steric hindrance.

More preferred substituents at the 38 position are more flexible, and/or smaller, and include alanine, glycine, leucine, isoleucine, valine, asparagine, serine and threonine. As with substituents in other positions, non-standard amino acids may also be used, such as α- aminobutyric acid. Further examples of non-standard amino acids are given in Table 2, although these are not necessarily limiting, as the skilled person will understand which amino acids will be appropriate, given the stearic and polar/non-polar requirements given by the present application. Where such substituents provide no advantage, either in stereoselectivity or catalytic activity, then these are generally not preferred, other than where it is possible to encode them in the genetic sequence of a host suitable to express the enzyme. Where the enzyme of the invention is synthesised by means other than straightforward expression by an engineered host, the feasibility of using unusual amino acids increases, and may be preferred.

It is particularly preferred that such enzymes be robust, in that they are capable of selective oxygenation of a substrate in the presence of hydrogen peroxide without the need for ancillary enzymes. In a preferred embodiment, the provision of a low spin enzyme, carrying the additional N70H mutation (in addition to the three in the cavity), further facilitates stability of the enzyme under continuous turnover conditions.

While it is generally preferred that the enzyme of the present invention is derived from a robust molecule, and preferably from a robust enzyme, especially a haem-based peroxidase such as horseradish peroxidase, it is a particular advantage that the modified peroxidases, or peroxygenases, of the invention have activity similar to that of cytochrome P450 or chloroperoxidases, whilst also being capable of acting without the need for the presence of ancillary enzymes. In particular, especially with the preferred embodiments of the invention, sub-micromolar dissociation constants, indicating high specificity, for aromatic substrates that can be oxidised in a stereoselective manner are observed. Thus, the modified enzymes of the invention may be used as generic routes to oxidise substrates, and can find advantage in stereoselective synthesis, processing, or the resolution of chiral mixtures, for example. Such observed activities may further find use in carbon-hydrogen bond activation, stereoselective hydroxylation, epoxidation, synthesis, n-dealkylation and demethylation.

Figure 1 shows the structure of H42E:F41A:R38H refined to 1.8A

Figure 2: Determination of the dissociation constants for the HRP:CN:Ferulate complex by IH NMR. K_D WT = 3.7 +/- 0.3mM; K_D A140G = 4.2 +/- 0.3mM; K_D A140Q > 2OmM. Figure 2A shows the Ferulaic acid concentration (mM) against values for (-δHz) Cl 8₃ (vertical axis) for the wildtype (WT) and the A140G mutant. Figure 2B shows the total ligand fitted against the "binding signal," constants K_D and (-δHz) C18₃ max floated. Fit is with f= (a)*((x+z+b)-((x+z+b)^Λ2-4*x*z)^Λ0.5)/(2*z). Fit f to y: x = Ferulate; y = (-5Hz) Cl 8₃; z = Enzyme Concentration at each titration point.

Figures 3 A and 3B show the introduction of a Rate Limiting Electron Transfer Step in the Al 4OQ mutant. Figure 3 A shows the concentration OfK₄Fe(CN)₆ in mM against K_obs in s^" ^l. Figure 3 B shows a schematic of the electron transfer step: A140Q CII + S <~K_D-> [A140Q CII ; S] ~K_ET~> A140Q RE + Products. K_D and KET were 5.58 +/- 0.44mM and 434 +/- 19 s^"1, resepectively.

Figure 4 shows the "third generation" of peroxygenase mimics. The previous best epoxidiser/CPO mimic was R38H:F41A:H42E" (HAE). The figure shows the positions of various amino acids including R38, F41, H42 and N70 used in the HAA, AAE, AAEH₅ AAAH.

Figure 5 shows the U V/ Vis properties of a number of the enzymes, including the wildtype and various mutants. Reference in the figure is made to (1): F Scheeider-Belhaddad, A.T. Smith et al (unpublished) and (2): H.A. Heering, A.T. Smith et al Biochem J. (2002) 363, p. 571-579.

Figure 6 shows that new variants containing a His at the N70 position were low spin but were also surprising active sulphoxidisers.

Figure 7 shows oxidation of thioanisole by open haem pocket mutants. The results shown in Figure 7A are represented graphically in Figure 7B, although CPO is not shown. The columns in &B are, from left to right, HRP (WT), F41 A, HAAE, HAA, AAE and AAEH.

Figure 8 shows the binding of aromatic substrates to the engineered cavity of AAEH and AAAH causes low spin to high spin conversion (MTN 2-(Methylthio)naphlene. TS: Thioanisole)

Figure 9 shows the enantioselectivity of catalysis using chiral HPLC to resolve the enantioselectivity of the sulphoxidation reaction.

Figure 10 shows that AAEH & AAAH exhibit non saturation kinetics with H₂O₂ presumably due to LS ligand. This results in an apparent 2ⁿ -order rate constant for peroxide activation in the presence of substrate. The graphical results were obtained under the following conditions: 10 mMphosphate/citrate buffer pH 7.0 @ 25 C 50 μMTS, 5% MeOH (v/V), thioanisole sulfoxidation at 254 nm. It is worth noting the following points: Sub μM binding for substrates; Spin state change on binding substrate activates en∑yme for reaction with peroxide; Enzyme remains active for hours at a time during steady state turnover; AAE and AAEH are essentially enantioselective; Active in 50% MeOH; Potent substrate inhibition.

E + S <- ► ES + H₂O₂ — ► Comp I:S + H₂O

E + P -«— ► EP + S <— ► EPS

Figure 11 shows an X-ray crystal from Figure 10. Figure 12 shows SEQ ID NO. 1 the amino acid sequence of the Horse Radish Peroxidase CIa isoenzyne.

Figure 13 shows sulphoxide oxidation by various enzyme variants in Coprinus cinereus. The AAE variant or mutant can be seen to be particularly effective.

Figure 14 shows that CiP mutants are beneficial too, especially the AAQ mutant is beneficial and the AAE mutant.

By robust it is meant that the enzyme is at least as stable as a cytochrome P450 enzyme and preferably more so, at a given temperature or under particular reaction conditions, such as normal cellular conditions, for instance salt concentrations, and temperature, such as 24 degrees C. It is also preferred that the stability is measured in 5OmM hydrogen peroxide, and that activity is stimulated in the presence of 20-50% methanol, as discussed below.

The enzyme may be derived from a cytochrome P450 or chloroperoxidase-type enzyme. It can be derived from peroxidases of fungal origin, preferably Basidiomycete, more preferably Coprinus sp and most preferably Coprinus cinereus. However, it is particularly preferred that the enzyme is derived from a plant, preferably a member of the Brassicaceae or leguminoceae family, more preferably a Horseradish peroxidase or soyabean peroxidase and most preferably from Armoracia rusticana (syn. Cochlearia armoracia) or Glycine max respectively. Suitable peroxidases may be determined by sequence homology analysis or may be as classified on the SCOP website, as discussed below.

The terms mutations and substitutions are used interchangeably herein, but it will be appreciated that they refer to changes made at said position when compared to the Horseradish Peroxidase (HRP), particularly that of SEQ ID NO.l. Whilst it may be the exact positional numbering of the amino acids may vary slightly, especially due to the presence of a terminal Met residue, for instance in the synthetic gene, post translational modifications or inter-species variation, it will be readily apparent to the skilled person how to asses which amino acids in the subject sequence correspond to, or are equivalent to, those of the named positions in SEQ ID NO. 1. This can often be done by eye, but sequence comparison programs such as BLAST, MEGALIG or CLUSTAL, may also be of use. In this case structural superposition to identify corresponding residues on the basis of conserved or matched residues conserved throughout the superfamily can be done, based on a backbone RMSD calculation using the molecular modelling programs QUANTA or INSIGHT II. RMSD is the Root Mean Square Deviation, calculated between Cα-atoms of matched residues at the best 3D superposition of the query and target structures.

The terms neutral or polar are standard in the art. Neutral refers to amino acids having side chains of no net charge. Standard non-polar residues include Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine and Tryptophan.

Polar side chains have portions that have a net positive (δ+) or negative charge (δ+) and the amino acid may be polar, but uncharged, such as Serine, Threonine, Asparagine, Glutamine, Tyrosine, or Cysteine, or polar and charged. In turn, the polar charged residues may be acidic, such as Aspartic acid or Glutamic acid, or basic, such as Lysine, Arginine or Histidine (positive).

By small or large amino acids, the skilled person will understand that this is reference to the steric bulk occupied by the side chain of a particular residue, when compared to other residues' side chains. This can be easily visualised by looking at the simple line structure of the residue, but 3-D models can also be consulted if necessary.

Preferred neutral residues for use in the enzymes of the invention, also referred to herein as modified enzymes, are alanine and glycine. Preferred polar residues are generally flexible, and include glutamic acid, aspartic acid, asparagine, glutamine, serine, threonine, lysine and arginine.

A particularly preferred neutral amino acid is alanine, and particularly preferred polar residues are those of glutamic acid and aspartic acid. Alanine is particularly preferred at position 38 and, optionally, 41, and preferably at both. Glutamic acid is particularly preferred at position 42, and although aspartic acid is less preferred, it is still capable of performing the desired function. Details of the full horseradish peroxidase gene sequence are given in Figure 2 of Smith, A.T., Santama, N., Dacey, S., Edwards, M., Bray, R.C., Thorneley, R.N.F. & Burke, J.F. (1990), "Expression of a synthetic gene for horseradish peroxidase in E. coli and folding

2+ and activation of the recombinant enzyme with Ca and heme", J. Biol. Chem. 265, 13335- 13343. The synthetic cloned wild type horseradish peroxidase sequence disclosed therein (SEQ ID NO. 2) further has an N-terminal Methionine, although this is not shown in SEQ ID NO.l. The sequence numbering is also that commonly used in the protein data bank (pdb) for the various X-rays structures of HRP, for example IATJ, NCBI Locus # AAA72223 (found for instance at http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=208494).

Meunier, B et al 1998 Biochem J v.330 ; Meno, K et al 2002 Acta Cryst. V.D58, of which one of the present inventors is a co-author address two related mutations for other reasons and no analysis of the oxygen transfer chemistry is made. In fact, these papers concerned different aspects of these mutants, such as structure, and mechanistic and redox properties, respectively.

By the term 'enzyme' is meant a biological molecule, preferably a protein, comprising a haem-based peroxidase catalytic centre of the invention. At its simplest, for example, the enzyme will correspond directly to horseradish peroxidase modified at least in positions 38, 41 and 42.

Preferably, the HRP (Horseradish Peroxidase) has the sequence of SEQ ID NO. 1. However, it will be appreciated that this is, in fact, the Horeseradish peroxidase CIa isoenzyme. At least 6 highly related genes exist in the horseradish while the average plant contains in excess of 90 peroxidase genes. All of these would be suitable for modification in a similar way to create the oxygen transfer catalysts analogous to those described herein. Essentially, the active site residues are conserved and isoenzyme variations are found outside of the active site residues considered here and are not immediately relevant to the invention, although the skilled person will appreciate that they may influence stability or expression levels in heterologous expression systems. The enzyme may also comprise an entirely different support structure, or may comprise another enzyme belonging to the plant peroxidase superfamily, provided that it is capable, of presenting the haem-based catalytic centre in such a way as to provide peroxygenase or peroxidase activity. It will be appreciated that such molecules may be mutants or variants of natural enzymes, or may be produced by genetic engineering or by selection by directed evolution using those described above as starting points. Mutants may be achieved by insertions, substitutions, inversions, deletions and duplications, as desired, while variants may be obtained from related species, for example. In general, it is preferred that the haem-based catalytic site of the enzyme of the invention be synthesised at the same time and with the remainder of the enzyme, in order to simplify the production process.

It is preferred that the enzyme has, or at least comprises, a portion containing the active site. Preferably, this includes the conserved 'plant peroxidase fold' of which SEQ ID NO. 1 is a classic, and preferred, example. Preferably, the enzyme is selected from the 'CCP- like family of haem-dependent peroxidases' group, as identified in the SCOP database, see for instance http://scop.berkeley.edU/data/scop.b.b.bec.b.b.html.

Although, overall, homology sequence levels for the plant peroxidase fold can, in some cases, be low, the overall fold is well classified within SCOP, as the 'CCP -like family of haem- dependent peroxidases' or is otherwise known as the 'plant peroxidase fold' referred to above. Thus, related homologues are easily identified.

However, it is preferred that the portion comprising the active site has at least 80% amino acid sequence homology to provided that the mutations of the invention and the oxygen transfer activity of the enzyme is retained. Preferably, the homology is at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, more preferably at least 99.5%, as appropriate. Suitable methods for assessing such homology are well known in the art, but may include use of the BLAST program or those discussed above, for instance. This can be tested by comparison of SEQ ID NOS. 1 and 15, for example.

Preferably the enzyme is produced by molecular biological methods, preferably by recombinant techniques as are well known in the art. It is a particular advantage of the present invention that the modified enzymes are capable of using hydrogen peroxide as a source of oxidising power and that activation of peroxide occurs- in a concerted manner after the binding of aromatic substrate, particularly in the low spin variants which contain the three active site mutations in addition to the N70H mutation.

Preferred products of the invention include the styrene epoxides, chiral sulphoxides, although this will be determined by the substituents that can be tolerated by the enzyme- mediated oxidation process. Preferably these include (methylsulphinyl)aryls, which by more complex dialkyl sulphoxides via sulphoxide-metal exchange, enantiomerically pure benzylsulphinyl(aryl) sulphoxide, which would allow the stereospecific incorporation of two new substituents and hence open a route to a diverse range of chiral sulphoxides. In particular, enantiomerically pure (methylsulphinyl)aryl sulphoxides are preferred as these make an excellent basis for the preparation of more complex chiral ligands. In their favour for this purpose, the current enzyme variants are fully active in 50% MeOH, give a 100% product yield with simple aromatic sulphides and maintain activity during turnover with 5OmM hydrogen peroxide over a period of hours. Additional suitable substrates for the enzymes include a diverse range of both activated and non-activated aromatic amines, sulphides and alcohols.

Within the plant peroxidase superfamily, the distal His (H42) and catalytic Arg (R38) (Fig. 1) are highly conserved and are known to play a major role in a general acid-base mechanism for the formation of compound I (c.f. accompanying Figure 13). Analysis of the X-ray structure of chloroperoxidase (CPO) does not reveal a His in the same location and yet the enzyme is capable of rapid compound I formation over a relatively wide pH range. Chloroperoxidase does, however, contain a GIu residue, slightly displaced toward the periphery of the active site (Fig 1). This GIu (El 83) is in turn hydrogen bonded to an adjacent His (H105) some distance from the haem iron. Chloroperoxidase is notable because of its extensive ability to catalyse oxygen transfer reactions from peroxide to a variety of aromatic compounds, which is also referred to as 'peroxygenase' activity. In contrast, the plant peroxidases have very limited ability in this direction. Site-directed mutants of HRP that permit deeper entry of aromatic substrates into the distal haem pocket have been shown to improve peroxygenase activity. Many of these mutations, however, severely compromise the ability of the enzyme to form and stabilise Compounds II and I because they involve key residues in the acid-base machinery of the enzyme, and specifically H42 and R38.

Without being bound by theory, it appears that substitution of the phenylalanine at position 41 and the arginine at 38 by smaller and/or more flexible residues, such as glycine or alanine, opens up the catalytic cleft. Further, substitution of the histidine at position 42 also opens the cleft but, in addition, it has further been found that the choice of residue, here, has a substantial effect on enantiomeric selectivity. The most preferred substitution so far known at position 42 is glutamate, especially when enantiomeric selectivity is required or preferred, as discussed below.

However, as Ala at position 42 promotes the level of overall activity but not enantioselectivity, it is preferred, especially if enantioselectivity is not required.

The overall effect, especially where the substitution at 42 is glutamate, is to allow the modified enzyme of the invention to accept the substrate directly into the cleft with sub- micromolar dissociation constants and often resulting in stereoselective oxidation, the latter most notably with glutamate at the 42 position.

It will be appreciated that substrate selectivity can be suitably engineered by appropriate selection of substituents in and around the catalytic cleft, particularly at the 38, 41 and 42 positions and, in a preferred embodiment additionally at the adjacent 70 position where His is beneficial to promoting a concerted reaction. It will be appreciated that these residues have a role in determining the area of the substrate molecule upon which the catalytic effect is directed, but that the structure of the remainder of the enzyme will determine the nature of the substrates with which the enzymes interact. Suitable scaffold molecules can readily be selected and/or designed by those skilled in the art.

As mentioned above, it is particularly preferred that position 42 be substituted by glutamic acid, where enantiomeric selectivity is required. It is preferred that position 38 be substituted by alanine for enantiomeric selectivity, but it would seem that considerably less selectivity is observed when position 42 is substituted by alanine, for example.

Preferred alternatives to Ala or GIu at position 42, especially when enantiomeric selectivity is required, are Ser, Thr, GIn and Asp, or other small, neutral residues.

Although GIu at position 42 is preferred when enantiomeric selectivity is required, it is not essential when sulphoxidation in general is required. For example, Ala works very well in this context and this would also be expected with other small neutral substitutions. Thus, the skilled person will be able to fine-tune the stereoselectivity of the enzyme of the present invention as required, with regard to the particular substrate required to be oxidised.

Surprisingly, it has also been established that substituting the Asparagine residue at position 70 with a histidine will generally have the effect of maintaining the catalytic site in a low-spin state until the substrate is bound, whereupon the low spin state undergoes a transition to a high spin state. This effect can be particularly advantageous in protecting the modified enzyme from hydrogen peroxide, for example, and this spin state transition is analogous to that seen when substrates bind to cytochromes P450. Without being bound by theory, it appears that the arginine in position 38 of the wild type prevents interaction of residue at position 70 with the haem pocket, and that the asparagine at position 70 in horseradish peroxidase would not interact with the haem pocket, even were the Arginine normally present in the wildtype at position 38 substituted by alanine. However, when alanine is present at position 38 and histidine at position 70, the histidine physically shifts to interact with the pocket and serves to force the haem iron into a low spin state. Particularly surprisingly, when a substrate binds in the engineered pocket, the haem iron reverts to the high spin state, and the catalytic peroxygenation can take place.

Thus, it is particularly preferred that the enzyme comprises alanine at position 38 and histidine at position 70.

Without being bound by theory, it is presently thought that only the deprotonated form in the imidazolate state works in this way. Although there are no other (standard) chemically equivalent residues, it is possible that Lys, Arg, Tyr or Trp may work as mutants at this position and are, hence, preferred, as these residues can ligate directly to Fe3+, at least. Indeed, this effect is particularly pronounced, as shown in accompanying Figure 10, which demonstrates that those enzymes of the invention that have low spin states (i.e. their active site haem groups have a low spin state) when not associated with substrate, i.e. those having a histidine at position 70, are resistant to high levels of hydrogen peroxide but never the less support good catalytic rates. Thus, those enzymes of the invention that remain in a high spin state at all times tend to have shorter life spans, as they are highly active in the presence of any amount of hydrogen peroxide, or other peroxide donor and, in addition, do not respond to increasing levels of peroxide. By contrast, those that undergo the P450 spin state transition are only active in the presence of substrate, so do not undergo self-inactivation by being active in the absence of substrate, and respond remarkably well to increasing levels of peroxide donor.

Accordingly, it will be appreciated that constitutively active enzymes will be useful in circumstances where an activation step is undesirable or inconvenient, and/or where it is desired that the enzyme only be active for a relatively short period. Those enzymes that undergo the T450-like' spin state transition will generally be more useful in industrial processes, or where longevity is an advantage.

Particularly preferred enzymes of the invention have the following substitutions: Enantiomeric selectivity R38A:F41A:H42E (AAE) SEQ ID NO.3

R38L:F41A:H42E SEQ ID NO.4

R38G:F41A:H42E SEQ ID NO.5 P450 Spin state transition R38A:F41A:H42A:N70H (AAAH) SEQ ID NO.6

R38H:F41A:H42A:N70H SEQ ID NO.7

R38A:F41G:H42A:N70H SEQ ID NO.8 Enantiomerically selective and P450 spin state transition

R38A:F41A:H42E:N70H (AAEH) SEQ ID NO.9

R38H:F41A:H42E:N70H SEQ ID NO.10

R38L:F41A:H42E:N7OH SEQ ID NO.ll

R38G:F41A:H42E:N70H SEQ ID NO.12

R38A:F41G:H42E:N70H SEQ ID NO.13

R38A:F41L:H42E:N70H SEQ ID N0.14 The oxidising power of the modified peroxidases of the present invention is preferably delivered by hydrogen peroxide, which is a commonly available, simple soluble oxidant. Without being bound by theory, the oxidising power appears to be exerted via a ferryl (Fe^IV=O prophyrin radical), Compound I-type nucleophillic intermediate of the haem-based catalytic centre of the modified enzyme, although an electrophillic Feπi -O-OH or Fem-O-O^" intermediate also cannot be excluded. If formation of one of these intermediates, probably Compound I occurs in the absence of bound aromatic substrate, then auto-oxidative damage to the enzyme, as discussed above, will ensue with or without reaction with a further hydrogen peroxide molecule. Without being bound by theory, the low spin form of the enzyme is unreactive to peroxide because the 6^th ligand position of the haem group is occupied by a nitrogenous (histidine 70) and or mixed water ligand (stabilised by glutamate 42 and histidine 70). This blocking ligand is displaced by the prior binding of an aromatic substrate and the enzyme converted to the active high spin state that only then reacts more rapidly with hydrogen peroxide. The enzyme is thus protected mechanistically from prior destruction by hydrogen peroxide.

All the above enzymes will be able to use alternate peroxides such as peracetic acid or metachloroperoxybenzoic acid in place of hydrogen peroxide and to do so may offer specific advantages for a given oxidation reaction. Thus, peracetic acid or metachloroperoxybenzoic acid are also preferred as substrates for sources of oxidising power.

Two horseradish peroxidase (HRP) variants, in particular, R38A:F41A:H42E (AAE) and R38A:F41A:H42E:N70H (AAEH), haven proven capable of 99-100% enantioselective sulphide oxidation. The presence of a distal GIu reside at the 42 position appeared instrumental for enantioselectivity as well as maintenance of a distal water molecule in the resting state of the enzyme, although other polar residues at this position will affect enantioselectivity in respect of different substrates.

A number of other variants, most notably R38A:F41A:H42A:N70H (AAAH), have, in addition to R38A:F41A:H42E:N70H (AAEH), also exhibited a substrate-dependent spin state transition normally seen only in cytochromes P450. All of these second-generation (quadruple) variants exhibited sub-micromolar dissociation constants for new substrates. The engineered haem cavity was shown to be highly effective in the catalysis of aromatic sulphide oxidation. By analogy, and as shown in accompanying Table 1 for certain horseradish peroxidase mutants, it is to be expected that these enzymes will be similarly effective in epoxidation and C-H bond activation.

The peroxygenases of the present invention provide unique tools for high value-added chemical synthesis. The enzymes of the present invention are capable of mass-production especially if transferred to the framework of Coprinus cinereus peroxidase currently in commercial production elsewhere. Recent results have shown that the sulphoxidation activity is directly transfered to Coprinus cinereus peroxidase when the corresponding positions are mutated, see for instance Figure 14. All of these variants, be they in horseradish peroxidase or Coprinus cinereus peroxidase or in any other member of the plant peroxidase fold family, offer a cheap means to achieve enantioselective production of drugs, for example, as they can be used for stereoselective synthesis, such as synthesis of chiral sulphoxides as standard reagents for chiral synthesis, and resolution of chiral mixtures. The enzymes of the present invention are also capable of C-H bond activation and stereoselective hydroxylation, N- dealkylation, and demethylation.

As shown in Example 2, Example 1 was repeated for corresponding mutants of Coprinus cinereus, using the same mutation short hand as for HRP. In other words, WT = wildtype; AAA = Alanine, Alanine, Alanine; AAE = Alanine, Alanine, Glutamate; and AAQ = Alanine, Alanine, Glutamine, wherein the mutants are at positions corresponding to positions 38, 41 and 42 of HRP (as per SEQ ID NO. 1 for instance). It can be seen that the AAQ mutant is beneficial and AAE mutant is particularly beneficial. In fact, in CiP, the residues corresponding to positions 38, 41 and 42 in HRP are 50, 53 and 54, respectively.

It is therefore preferred that the triple mutant positions in CiP are 50, 53 and 54, according to SEQ ID NO. 15. The NCBI locus is 1H3JB (found at http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=33356984 for instance). The synthetic gene sequence is covered in PCT/GB206/001515, incorporated herein by reference. Thus, when the enzyme is a fungal peroxidase, especially from Coprinus sp or Coprinus cinereus (also known as Coprinopsis cinerea), the mutations are preferably AAQ and most preferably AAE. However, quadruple mutants, especially at a position corresponding to N70 in HRP (e.g. SEQ ID NO. 1) are also envisaged and preferred.

The wildtype sequence of the CiP is given in SEQ ID NO. 15. The AAA, AAE and AAQ mutants are SEQ NOS. 16-18, respectively.

It is particularly advantageous that enzymatic turnover is stable in 5OmM hydrogen peroxide, and that activity is stimulated in the presence of 20-50% methanol, particularly in the low spin variants R38A:F41A:H42E:N70H and R38A:F41A:H42A:N70H, which are activated by the prior binding of substrate in a manner analogous to that of a cytochrome P450. This is likely to protect the enzyme mechanistically from inactivation by hydrogen peroxide at the level of Compound I.

It has been known for some time that substitutions in the distal haem pocket of HRP that open the haem cavity, can potentially enhance thioanisole oxidation and epoxidation reactions from essentially zero levels (for instance Savenoka, M et al 1996, (JBC 271). This has been shown for residue substitutions at positions 41 and 42 where the residue is small and hydrophobic. Although mutations at the 38 position have been made these have never been shown to enhance sulphoxidation / epoxidation (oxygen atom transfer). In the existing single or double mutants (41 and 42 position Ala mutants), the levels of activity achieved are very low, (generally < 1.0 s^"1) and the reactions catalysed are not enantioselective. Furthermore, the variants are very readily inactivated by hydrogen peroxide.

We had earlier (Meno et al) studied the ability of other weak bases eg GIu to activate peroxide when placed at position 42. However, this publication makes no mention of their surprising ability to sulphoxidise or expoxidate when combined with substitutions at the 41 or 38 positions, hi particular, no mention of triple mutants involving the addition of the R38A mutation, showing enhanced oxygen transfer chemistry in its own right, is made. Further a combination of all three in which no surrogate His is present in the haem cavity, would not have been expected to activate hydrogen peroxide fast enough to be useful, but has a number of unpredicted and very useful effects, especially when combined with an additional mutation, N70H outside the haem pocket.

These advantages include:

• The inclusion of at GIu residue at the 42 position as part of a combination of the three changes is important to maintain the enantioselectivity of the engineered activity (essentially 100% enantiomeric excess (e.e.)).

• The rates achieved are nearly 100 times better that any previous mutations.

• The engineered catalysts are resistant to inactivation in the presence of substrate and the yields of product are similarly essentially 100%.

• The triple mutant variants (AAE, AAEH and) exhibit tight sub micromolar dissociation constants for a range of non natural substrates.

• Variants containing an additional His at the 70 position (AAEH and AAAH) are even more resistant to inactivation by hydrogen peroxide because they exhibit a substrate induced spin state change analogous to that seen in cytochrome P450's on substrate binding, which activates the enzyme for reaction with hydrogen peroxide. The mechanistic protection of the enzyme from hydrogen peroxide inactivation could not have been predicted.

Thus, the catalytic rates achieved in particular by AAE and AAEH are superior to those achieved with any previous HRP or myoglobin mutants designed for this purpose. The rate of oxygen atom transfer is substantially faster than the formation of peroxidase Compound I in the absence of substrate, suggesting a concerted mechanism in which prior binding of substrate activates (by removing the low spin ligand) the enzyme for reaction with hydrogen peroxide. These new catalysts can activate oxygen directly under appropriate conditions and enzymes of this type may offer a cleaner and more efficient route for making ingredients that are required for the synthesis of important drugs and biomolecules in a more environmentally-friendly manner than previously. Most importantly, they could serve as templates for further selection, using forced evolution and homologous recombination techniques in conjunction with substrate panel screens, for synthetic designer enzymes tuned for a particular chemical synthesis, for high value-added product synthesis that is difficult to achieve by conventional synthetic strategies.

The modified enzymes of the present invention are preferably based on horseradish peroxidase, but may also be based on any commercially available peroxidase enzyme, such as Coprinus cinereus peroxidase, and it will be appreciated that the person skilled in the art will readily be able to establish the residues in such alternative peroxidases that correspond to residues 38, 41, 42 and 70 of horseradish peroxidase. Coprinus cinereus peroxidase and other members of the plant peroxidase superfamily family may be determined by sequence homology analysis, as discussed above, or may be as classified on the SCOP (Structural Classification of Proteins) database provided by Berkeley University and available, for instance at http://scop.berkeley.edu

In fact, in a recent PCT filing PCT/GB2006/001515 (incorporated herein by reference) we detailed mutations in Coprinus Cinereus peroxidase (CiP) produced commercially by Novozymes which in part create the activity of lignin peroxidase. Using the same synthetic gene described we have tested a sub selection of the residue positions described herein, most notably the equivalent of AAE. This also shows evidence of direct oxygen transfer activity showing that the properties of the horseradish peroxidase mutants of the present invention are transferable to other peroxidases of fungal origin.

In other words, the functional effects of the mutations are likely to be generic. Thus, if residue substitutions are made at the corresponding residue positions of other peroxidases, they are also likely to become oxygen transfer catalysts. Thus, CiP is also a preferred enzyme of the invention, once mutated as discussed herein, particularly as it is in commercial production at g/1 levels from a recombinant source (Novozymes) and is therefore already an established vehicle to exploit the present invention. However, it is worth pointing out that horseradish peroxidase is thought to retain superior stability characteristics and thus is especially preferred. Also provided is the use of the enzyme in a stereoselective synthetic process, in the presence of 0-100% methanol, preferably 20-50% methanol. The invention also provides a method of oxygen transfer, catalysed by the enzyme.

The present invention will now be further illustrated with respect to the following, non-limiting Examples.

EXAMPLE 1

Construction of horseradish peroxidase mutants with selective oxygenation activities similar to those of cytochromes P450 and chloroperoxidase

Crystallography of the HRP mutants

We refined H42E:R38H and crystallised H42E:F41A:R38H and refined the structure to 1.8A (Figure 1). Both mutants crystallised in the normal P2₁2₁2₁ space group and were able to oxidise styrene. R38H:F41A:H42E (HAE) had been identified as the best styrene epoxidiser. Both structures showed more open distal haem cavities. Interestingly, E42 appeared to be pulled up out of the haem pocket due to H-bond formation with N70, so mimicking the connection that is formed between H42 in and N70 in the WT structure. In part, this contributed to the more open pocket. These earlier mutants were subsequently evaluated for their sulphoxidation activity with aromatic sulphides.

Crystal structures of hyperactive mutants

Previously the hyperactive peroxidase mutants A140G (IGWO) and its comparator A140Q (IGWU) had been constructed and characterised. The GIn side chain of the A140Q variant mutant was shown to hinder the haem-edge substrate access channel as predicted, accounting for its low activity (1%) compared to WT with most small phenolic substrates. NMR binding studies confirmed that A140Q hardly interacted with substrates at all compared to WT (see Figure 2) and resulted in a detectable rate-limiting electron transfer step during the reduction of Compound II, under stopped flow conditions (Figure 3). The A140G mutant had a significantly more open haem-edge substrate access channel and was up to two times more active with most substrates. Detailed steady state and pre steady state analysis of both variants showed that this was directly the result of an increase in the rate determining electron transfer step (data not shown).

Design and characterisation of chloroperoxidase / P450 / peroxygenase functional mimics

The insights provided by the structures above confirmed the presence of a more open cavity not normally seen in the WT. Stopped flow studies also confirmed that the peroxidase intermediates were still generated but were unstable and, therefore, presumably more reactive than in the WT. We decided to go one step further and extend the size of the distal cavity by removing the surrogate His from the cavity entirely but retaining the GIu residue at position 42, as this seemed, in the light of the new structures, to be consistent with an open cavity, while providing at least some potential for a weak base to activate peroxide. We therefore generated some 5 new variants; these are listed in Table 1 together with earlier chloroperoxidase mimics. In some of the new variants we introduced an additional His at the N70 position in an attempt to create a H-bond interaction between E42 and H70 as seen in chloroperoxidase. The UV/Vis properties of these and some of the older variants are shown in Figure 5.

Interestingly, all variants remained high spin (HS) unless the haem pocket was both 'open' and a His was present at position 70. Only AAEH and AAAH (see short hand mutant coding system in Table 1) were low spin at pH 7.0, but became HS at pH 5 (see helium EPR and UV/Vis spectra in Figure 6), presumably driven by the protonation of His 70. The EPR results depicted in Figure 5 are indicative of mixed low spin nitrogenous and low spin water ligation at pH 7.0 for AAEH while, for the AAAH variant, there appears to be a pure low spin nitrogenous ligation at pH 7.0 (EPR data not shown). The results are indicative of a major conformational rearrangement (3-4A) of the B-C loop region containing His70 as it moves down into direct ligation distance of the haem iron. This re-arrangement is thought to be caused by the removal of the underlying catalytic Arg (R38), which is normally in direct Van der Waals contact with the residue at position 70. Despite the apparent low spin nature of AAEH and AAAH (the 6^th co-ordination position should be free to react with peroxide) they are still excellent oxidisers of aromatic sulphides (Figure 7) with k_cat values in excess of 2-10 s^'1 and sub micromolar Km values for thioanisole. These reactions were not catalysed by the wild-type (WT) enzyme at all.

In Figure 8, the reason for this becomes apparent. The addition of substrates at pH 7.0 (thioanisole and,/ or 2-(methylthio)naphthalene) causes a LS to HS spin state transition. It is complete and can be monitored in the UV/Vis to determine the substrate dissociation constants, all of which are in the low micromolar range (Table in Figure 8). Even the largely high spin variant AAE shows sufficient change from a 6cHS to 5cHS form on addition of substrate, from which substrate dissociation constants can be determined in a similar way (see Figure 7). Careful analysis of the binding curves for smaller substrates such as thioanisole reveals the presence of two binding sites, one high affinity and one low. It seems probable that the low affinity site in the traditional haem-edge peroxidase substrate interaction site is centred on F 179 and the haem Cl 8 methyl, while the high affinity site represents binding of aromatic sulphide to the engineered cavity above the haem iron.

Figure 9 shows the resolution of the products of thioanisole oxidation by chiral HPLC. Only the S sulphoxide enantiomer (100% pure) is produced during catalysis by AAE and AAEH, just as with chloroperoxidase, strongly implying a direct oxygen atom transfer mechanism. This enantioselectivity is also analogous to that seen in P450-type systems. Interestingly, the catalysis of AAAH (which has the pure nitrogenous low spin ligand, presumably His 70) is not similarly enantioselective, implying that E42 is both important for the retention of a low spin distal water molecule in the AAE and AAEH variants and for the enantioselectivity of catalysis. The substrate induced spin state transition seen on addition of substrate is reminiscent of P450 behaviour, in which the aromatic substrate binding excludes a water molecule from the haem active site. Additional kinetic data for some of the mutants of the invention is summarised in Figure 10. The mechanistic situation is complex and only limited pre-steady state analysis of these mutants has been undertaken. The low spin mutants show a second order dependence on hydrogen peroxide for catalysis of sulphoxidation, implying a slow rate limiting reaction with peroxide presumably to form a Compound Hike state. Spectra characteristic of this intermediate have been observed under pre-steady state conditions in the absence of the sulphide substrate for both AAE and AAEH. Reaction of a preformed AAE: thioanisole complex with peroxide under stopped flow conditions, in which the loss of substrate is followed together with rapid scan spectra of the haem, shows a classic burst phase in thioanisole consumption, without detectable accumulation of Compound I. This implies that the concerted 2-e oxygen atom transfer reaction is much faster than Compound I formation. Under steady state turnover conditions the more open haem pocket variants are subject to a potent substrate inhibition (Figure 7) (reaction scheme described in description of Figure 10 and used to model the data in Figure 7) in which the binding of substrate at the second low affinity site may block the release of product (a form of non competitive substrate inhibition).

Styrene epoxidation and aromatic sulphide oxidation

Unlike the wild type, all three mutants and H42E were able to epoxidate styrene directly (Table 3). Most notably, H42E:F41A:R38H shows a dramatic increase in styrene epoxidation activity. The activity is stable even at high hydrogen peroxide concentrations and unlike chloroperoxidase is not suppressed by chloride ions. All are able to form compounds I and II although in the case of H42E:F41A:R38H the lifetime of compound I is limited to 10- 20sec and can only be detected in stopped-flow rapid-scan experiments. Clearly the reactivity of the intermediates formed is enhanced.

Table 1 shows styrene epoxidation by HRP mutants. Specific activity measurements

were made at saturating concentrations of styrene for different mutant enzymes. Reaction products were extracted from samples taken at different time points and analysed by GC-MS. Measurements were started by the addition of enzyme in a reaction mixture containing 10 mM H₂O₂, 5 mM styrene in 100 mM sodium phosphate buffer pH=7.0. Table 1

Enzyme Styrene Phenylacetal- ratio epoxide dehyde SO/PAA

HRP-WT 0.01 tr ^a -

H42E 7.4 10.6 0.7

H42E:R38H 12 8.4 1.42

H42E:F41A:R38H 126 257 0.5

H42E:F41H:R38A 40 61 0.65

EXAMPLE 2

Example 1 was repeated for corresponding mutants of Coprinus cinereus, using the same mutation short hand as for HRP. hi other words, WT = wildtype; AAA = Alanine, Alanine, Alanine; AAE = Alanine, Alanine, Glutamate; and AAQ = Alanine, Alanine, Glutamine, wherein the mutants are at positions corresponding to positions 38, 41 and 42 of HRP (as per SEQ ID NO. 1 for instance). It can be seen from Figure 14 that the AAQ mutant is beneficial and AAE mutant is particularly preferred.

The CiP R51A:F54A:H55E mutant (which corresponds to positions 50, 53 and 54 in SEQ ID NOS. 16-18 which lack a terminal Met) was encoded at the DNA level using synthetic nucleotides in a standard PCR based mutagenesis procedure as described in Doyle et al 1998 (Doyle, W. A., Blodig, W., Veitch., N., Piontek, K., & Smith A.T. (1998) Two substrate interaction sites in lignin peroxidase revealed by site directed mutagenesis. Biochemistry 37, 15097-15105). Recombinant protein was expressed and refolded as described in PCT/GB206/001515. Standard sulphoxidation assays were conducted in exactly the same way as for the HRP mutants.

Table 2 : non-standard amino acids

Claims

Claims:

1. A haem-based peroxygenase enzyme, wherein positions R38, F41 and H42, by comparison with accompanying SEQ ID NO. 1, are substituted, and wherein the arginine at position 38 is substituted by a smaller residue, the phenylalanine at position 41 is substituted by a smaller, neutral residue and the histidine at position 42 is substituted by another polar, or neutral residue.

2. An enzyme according to claim 1, wherein the histidine at position 42 is substituted by an acidic residue.

3. An enzyme according to claim 1 or 2, wherein asparagine at position 70 by comparison with accompanying SEQ ID NO. 1 is replaced by histidine (N70H).

4. An enzyme according to any preceding claim, wherein 38 position is substituted by alanine, glycine, leucine, isoleucine, valine, asparagine, serine, threonine or α- aminobutyric acid.

5. An enzyme according to any preceding claim, derived from a cytochrome P450 or chloroperoxidase-type enzyme.

6. An enzyme according to any of claims 1-4, derived from peroxidases of fungal origin

7. An enzyme according to claim 6, wherein the fungus is a Basidiomycete.

8. An enzyme according to claim 7, wherein the fungus is a derived from Coprinus sp.

9. An enzyme according to claim 8, wherein the fungus is Coprinus cinereus.

10. An enzyme according to any of claims 1-4, wherein the fungus is derived from a plant.

11. An enzyme according to claim 10, which is derived from the Brassicaceae or leguminoceae family.

12. An enzyme according to claim 11, which is derived from a Horseradish peroxidase or soyabean peroxidase.

13. An enzyme according to claim 12, which is derived from Armor acia rusticana (syn. Cochlearia armoracia) or Glycine max.

14. An enzyme according to any of claims 1-5 and 10-13, derived from members of the plant peroxidase superfamily family as classified on the SCOP database.

15. An enzyme according to any preceding claim, wherein the neutral residue is selected from the group consisting of: Glycine, Alanine, Valine, Leucine, Isoleucine, Methionine, Proline, Phenylalanine and Tryptophan.

16. An enzyme according to any preceding claim, wherein the polar side chains are polar, but uncharged.

17 An enzyme according to claim 16, wherein the polar side chains are selected from the group consisting of: as Serine, Threonine, Asparagine, Glutamine, Tyrosine, or Cysteine.

18. An enzyme according to any preceding claim, wherein the polar side chains are acidic, such as Aspartic acid or Glutamic acid.

19. An enzyme according to any preceding claim wherein the polar side chains are basic, such as Lysine, Arginine or Histidine.

20. An enzyme according to claim 15, wherein neutral residues are alanine or glycine.

21. An enzyme according to any preceding claim, wherein position 42 is replaced by glutamic acid.

22. An enzyme according to any preceding claim, wherein position 38 is substituted by one of alanine, glycine, leucine, isoleucine, valine, serine and threonine.

23. An enzyme according to claim 22, wherein position 38 and, optionally, 41, is replaced by alanine.

24. An enzyme according to any preceding claim, wherein the modified enzyme comprises alanine at position 38 and histidine at position 70, by comparison with SEQ ID NO. 1.

25. An enzyme according to any of claims 1-13, wherein the residues at positions corresponding to positions 38, 41 and 42 in SEQ ID NO.l are A, A, and E, respectively.

26. An enzyme according to any of claims 1-13, wherein the residues at positions corresponding to positions 38, 41, 42 and 70 in SEQ ID NO.l are A, A, E₅ and H, respectively.

27. An enzyme according to any of claims 1-13, wherein the residues at positions corresponding to positions 38, 41, 42 and 70 in SEQ ID NO.l are A, A, A, and H, respectively.

28. An enzyme according to any of claims 24-27, having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.l l, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, whilst still retaining the residues specified at positions corresponding to positions 38, 41 and 42 and, optionally, 70, of SEQ ID NO. 1.

29. An enzyme according to any of claims 1-9, wherein the residues at positions corresponding to positions 38, 41 and 42 in SEQ ID NO.l are A, A, and E, respectively.

30. An enzyme according to claim 29, wherein the residues correspond to positions 50, 53 and 54 in SEQ ID NO.15.

31. An enzyme according to any preceding claim, wherein the modified enzyme is capable of using hydrogen peroxide as a source of oxidising power.

32. An enzyme according to any preceding claim, wherein the enzyme is capable of catalysing the production of styrene epoxides, chiral sulphoxides, (methylsulphinyl)aryls, enantiomerically pure benzylsulphinyl(aryl) sulphoxide and activated and non-activated aromatic amines, sulphides and alcohols.

33. Use of an enzyme according to any preceding claim in a stereoselective synthetic process, in the presence of 0-100% methanol, preferably 20-50% methanol.

34. A method of oxygen transfer, catalysed by an enzyme according to any of claims 1-32 or as defined in claim 33.