HEMOPROTEIN CATALYSTS FOR IMPROVED ENANTIOSELECTIVE ENZYMATIC SYNTHESIS OF TICAGRELOR
20180148745 ยท 2018-05-31
Assignee
Inventors
- Francis H. Arnold (La Canada, CA, US)
- Hans Renata (Pasadena, CA, US)
- John A. McIntosh (Pasadena, CA, US)
- Russell D. Lewis (Pasadena, CA)
- Sek Bik Jennifer Kan (Pasadena, CA)
- Kari Hernandez (Pasadena, CA, US)
- Pedro Coelho (Santa Monica, CA)
- David Rozzell (Santa Monica, CA, US)
- Chen Zhang (Santa Monica, CA, US)
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12Y106/02004
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides methods by which trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine and related cyclopropane compounds are prepared using synthetic strategies that include a biocatalytic cyclopropanation step.
Claims
1. A reaction mixture for producing a cyclopropanation product of Formula A: ##STR00049## wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, C.sub.1-18 alkynyl, C.sub.1-18 alkoxy, C.sub.1-18 alkenyloxy, C.sub.1-18 alkynyloxy; and the reaction mixture comprises an olefinic substrate, a carbene precursor, and a heme enzyme.
2. The reaction mixture of claim 1, wherein R.sup.6 is C.sub.1-18 alkoxy and the carbene precursor is a diazoester.
3. The reaction mixture of claim 2, wherein the cyclopropanation product is a compound of Formula XVII: ##STR00050## the olefinic substrate is a compound of Formula V ##STR00051## and the carbene precursor is a compound of Formula XVI, ##STR00052## wherein R.sup.6a is C.sub.1-18 alkyl.
4. The reaction mixture of claim 3, wherein the cyclopropanation product is a compound according to Formula XVIIa: ##STR00053##
5. The reaction mixture of claim 3, wherein the cyclopropanation product is a compound according to Formula VIIa: ##STR00054##
6. The reaction mixture of claim 1, wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, and C.sub.1-18 alkynyl and the carbene precursor is a diazoketone.
7. The reaction mixture of claim 6, wherein the cyclopropanation product is a compound of Formula XXVII: ##STR00055## the olefinic substrate is a compound of Formula V: ##STR00056## and the carbene precursor is a compound of Formula XXVI: ##STR00057## wherein R.sup.6b is C.sub.1-18 alkyl.
8. The reaction mixture of claim 7, wherein the cyclopropanation product is a compound according to Formula XXVIIa: ##STR00058##
9. The reaction mixture of claim 8, wherein R.sup.6b is methyl.
10. The reaction mixture of any one of the preceding claims, wherein the heme enzyme comprises a mutation at the axial position of the heme coordination site.
11. The reaction mixture of any one of the preceding claims, wherein the heme enzyme is a cytochrome P450 enzyme or a variant or homolog thereof.
12. The reaction mixture of claim 11, wherein the cytochrome P450 enzyme is a P450 BM3 enzyme or a variant or homolog thereof.
13. The reaction mixture of claim 11, wherein the cytochrome P450 enzyme is a CYP119 enzyme or a variant or homolog thereof.
14. The reaction mixture of claim 11, wherein the cytochrome P450 enzyme is a CYP119 variant or homolog encoding a mutation at position H315 to any other amino acid, for example alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.
15. The reaction mixture of any one of the preceding claims, wherein the heme enzyme is a cytochrome c or a variant or homolog thereof.
16. The reaction mixture of claim 1, wherein the heme enzyme is a globin or a variant or homolog thereof.
17. The reaction mixture of any one of claims 2-10, wherein the heme enzyme is a globin or a variant or homolog thereof.
18. The reaction mixture of claim 16, wherein the heme enzyme is a myoglobin or a variant or homolog thereof.
19. The reaction mixture of claim 16, wherein the globin is M. infernorum hemoglobin according to SEQ ID NO: 61 or a variant or homolog thereof.
20. The reaction mixture of claim 19, wherein the M. infernorum hemoglobin variant or homolog comprises one or more mutations of amino acid residues selected from the group consisting of F28, Y29, L32, L54, and V95.
21. The reaction mixture of claim 19, wherein the M. infernorum hemoglobin variant or homolog comprises one or more mutations selected from the group consisting of F28S, Y29A, L32A, L32C, L32T, L54S, and V95F.
22. The reaction mixture of claim 21, wherein the M. infernorum variant or homolog comprises a V95F mutation.
23. The reaction mixture of claim 16, wherein the globin is B. subtilis truncated hemoglobin according to SEQ ID NO: 62 or a variant or homolog thereof.
24. The reaction mixture of claim 23, where the B. subtilis hemoglobin variant or homolog comprises one or more mutations of amino acid residues selected from the group consisting of T45 and Q49.
25. The reaction mixture of claim 24, where the B. subtilis hemoglobin variant or homolog comprises a T45 mutation and a Q49 mutation.
26. The reaction mixture of claim 23, where the B. subtilis hemoglobin variant or homolog comprises one or more mutations selected from the group consisting of T45L, T45F, T45A, Q49L, Q49F, and Q49A.
27. The reaction mixture of claim 26, where the B. subtilis hemoglobin variant or homolog comprises a first mutation selected from the group consisting of T45L, T45F, and T45A, and a second mutation selected from the group consisting of Q49L, Q49F, and Q49A.
28. The reaction mixture of any one of the preceding claims, wherein the cyclopropanation product is produced in vitro.
29. The reaction mixture of any one of the preceding claims, wherein the reaction mixture further comprises a reducing agent.
30. The reaction mixture of any one of the preceding claims, wherein the heme enzyme is localized within a whole cell and the cyclopropanation product is produced in vivo.
31. The reaction mixture of any one of the preceding claims, wherein the cyclopropanation product is produced under anaerobic conditions.
32. A method for producing a cyclopropanation product of Formula A: ##STR00059## wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, C.sub.1-18 alkynyl, C.sub.1-18 alkoxy, C.sub.1-18 alkenyloxy, C.sub.1-18 alkynyloxy; the method comprising forming a reaction mixture containing an olefinic substrate, a carbene precursor, and a heme enzyme under conditions sufficient to form the cyclopropanation product.
33. The method of claim 32, wherein R.sup.6 is C.sub.1-18 alkoxy and the carbene precursor is a diazoester.
34. The method of claim 33, wherein the cyclopropanation product is a compound of Formula XVII: ##STR00060## the olefinic substrate is a compound of Formula V ##STR00061## and the carbene precursor is a compound of Formula XVI, ##STR00062## wherein R.sup.6a is C.sub.1-18 alkyl.
35. The method of claim 34, wherein the cyclopropanation product is a compound according to Formula XVIIa: ##STR00063##
36. The method of claim 34, wherein the cyclopropanation product is a compound according to Formula VIIa: ##STR00064##
37. The method of claim 32, wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, and C.sub.1-18 alkynyl and the carbene precursor is a diazoketone.
38. The method of claim 37, wherein the cyclopropanation product is a compound of Formula XXVII: ##STR00065## the olefinic substrate is a compound of Formula V: ##STR00066## and the carbene precursor is a compound of Formula XXVI: ##STR00067## wherein R.sup.6b is C.sub.1-18 alkyl.
39. The method of claim 38, wherein the cyclopropanation product is a compound according to Formula XXVIIa: ##STR00068##
40. The method of claim 39, wherein R.sup.6b is methyl.
41. The method of any one of claims 32-39, wherein the heme enzyme comprises a mutation at the axial position of the heme coordination site.
42. The method of any one of claims 32-41, wherein the heme enzyme is a cytochrome P450 enzyme or a variant thereof.
43. The method of claim 42, wherein the cytochrome P450 enzyme is a P450 BM3 enzyme or a variant thereof.
44. The method of claim 42, wherein the cytochrome P450 enzyme is a CYP119 enzyme or a variant thereof.
45. The method of claim 42, wherein the cytochrome P450 enzyme is a CYP119 variant encoding a mutation at position H315 to any other amino acid, for example alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.
46. The method of any one of claims 32-41, wherein the heme enzyme is a cytochrome c or a variant thereof.
47. The method of claim 32, wherein the heme enzyme is a globin or a variant thereof.
48. The method of any one of claims 33-41, wherein the heme enzyme is a globin or a variant thereof.
49. The method of claim 47, wherein the heme enzyme is a myoglobin or a variant thereof.
50. The method of claim 47, wherein the globin is M. infernorum hemoglobin according to SEQ ID NO: 61 or a variant thereof.
51. The method of claim 50, wherein the M. infernorum hemoglobin variant comprises one or more mutations of amino acid residues selected from the group consisting of F28, Y29, L32, L54, and V95.
52. The method of claim 50, wherein the M. infernorum hemoglobin variant comprises one or more mutations selected from the group consisting of F28S, Y29A, L32A, L32C, L32T, L54S, and V95F.
53. The method of claim 50, wherein the M. infernorum variant comprises a V95F mutation.
54. The method of claim 47, wherein the globin is B. subtilis truncated hemoglobin according to SEQ ID NO: 62 or a variant thereof.
55. The method of claim 54, where the B. subtilis hemoglobin variant comprises one or more mutations of amino acid residues selected from the group consisting of T45 and Q49.
56. The method of claim 55, where the B. subtilis hemoglobin variant comprises a T45 mutation and a Q49 mutation.
57. The method of claim 54, where the B. subtilis hemoglobin variant comprises one or more mutations selected from the group consisting of T45L, T45F, T45A, Q49L, Q49F, and Q49A.
58. The method of claim 54, where the B. subtilis hemoglobin variant comprises a first mutation selected from the group consisting of T45L, T45F, and T45A, and a second mutation selected from the group consisting of Q49L, Q49F, and Q49A.
59. The method of any one of claims 32-58, wherein the cyclopropanation product is produced in vitro.
60. The method of any one of claims 32-59, wherein the reaction mixture further comprises a reducing agent.
61. The method of any one of claims 32-60, wherein the heme enzyme is localized within a whole cell and the cyclopropanation product is produced in vivo.
62. The method of any one of claims 32-61, wherein the cyclopropanation product is produced under anaerobic conditions.
63. The method of any one of claims 32-62, further comprising converting the cyclopropanation product to ticagrelor.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0035]
[0036]
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0037] The present disclosure relates to novel methods for the preparation of phenylcyclopropylamine derivatives, which are useful intermediates in the preparation of triazolo [4,5-d]pyrimidine compounds. The present disclosure particularly relates to novel, commercially viable and industrially advantageous methods for the preparation of a substantially pure ticagrelor intermediate, trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine. The intermediate is useful for preparing ticagrelor, or a pharmaceutically acceptable salt thereof, in high yield and purity. Because the biocatalytic cyclopropanation route uses an enzyme to set the chirality of the cyclopropane, the method of the present invention obviates the need to use chiral auxiliaries or chromatographic separation. Additionally, the method described herein requires only simple reagents, thus affording the cyclopropane product at a lower cost than published alternatives.
[0038] One of the published methods for making trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine using conventional chemistry methods is shown in Scheme 6. See, Owen et al., Drugs Fut., 32(10):845-853 (2007). This method for making trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine employs conventional chemistry that requires seven steps. In contrast, the present invention provides a low-cost and environmentally friendly synthesis of this key cyclopropane intermediate of ticagrelor by advantageously reducing the number of synthetic steps from 7 to 2, thus offering a substantial economic advantage.
##STR00009##
II. Definitions
[0039] The following definitions and abbreviations are to be used for the interpretation of the invention. The term invention or present invention as used herein is a non-limiting term and is not intended to refer to any single embodiment but encompasses all possible embodiments.
[0040] As used herein, the terms comprises, comprising, includes, including, has, having, contains, containing, or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, or refers to an inclusive or and not to an exclusive or.
[0041] The terms about and around, as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If X were the value, about X or around X would indicate a value from 0.9X to 1.TX, and more preferably, a value from 0.95X to 1.05X. Any reference to about X or around X specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, about X and around X are intended to teach and provide written description support for a claim limitation of, e.g., 0.98X.
[0042] The term cyclopropanation (enzyme) catalyst or enzyme with cyclopropanation activity refers to any and all chemical processes catalyzed by enzymes, by which substrates containing at least one carbon-carbon double bond can be converted into cyclopropane products by using diazo reagents as carbene precursors.
[0043] The terms engineered heme enzyme and heme enzyme variant include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.
[0044] The terms engineered cytochrome P450 and cytochrome P450 variant include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes.
[0045] The term whole cell catalyst includes microbial cells expressing heme-containing enzymes, wherein the whole cell catalyst displays cyclopropanation activity.
[0046] As used herein, the terms porphyrin and metal-substituted porphyrins include any porphyrin that can be bound by a heme enzyme or variant thereof. In particular embodiments, these porphyrins may contain metals including, but not limited to, Fe, Mn, Co, Cu, Rh, and Ru.
[0047] The terms carbene equivalent and carbene precursor include molecules that can be decomposed in the presence of metal (or enzyme) catalysts to structures that contain at least one divalent carbon with only 6 valence shell electrons and that can be transferred to CC bonds to form cyclopropanes or to CH or heteroatom-H bonds to form various carbon ligated products.
[0048] The terms carbene transfer and formal carbene transfer as used herein include any chemical transformation where carbene equivalents are added to CC bonds, carbon-heteroatom double bonds or inserted into CH or heteroatom-H substrates.
[0049] As used herein, the terms microbial, microbial organism and microorganism include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.
[0050] As used herein, the term non-naturally occurring, when used in reference to a microbial organism or enzyme activity of the invention, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the cyclopropanation activity described above.
[0051] As used herein, the term anaerobic, when used in reference to a reaction, culture or growth condition, is intended to mean that the concentration of oxygen is less than about M, preferably less than about 5 M, and even more preferably less than 1 M. The term is also intended to include sealed chambers of liquid or solid medium maintained with an atmosphere of less than about 1% oxygen. Preferably, anaerobic conditions are achieved by sparging a reaction mixture with an inert gas such as nitrogen or argon.
[0052] As used herein, the term exogenous is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.
[0053] The term heterologous as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.
[0054] On the other hand, the term native or endogenous as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicates molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.
[0055] The term homolog, as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
[0056] A protein has homology or is homologous to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have similar amino acid sequences. Thus, the term homologous proteins is intended to mean that the two proteins have similar amino acid sequences. In particular embodiments, the homology between two proteins is indicative of its shared ancestry, related by evolution.
[0057] The terms analog and analogous include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.
[0058] As used herein, the term alkyl refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C.sub.1-2, C.sub.1-3, C.sub.1-4, C.sub.1-5, C.sub.1-6, C.sub.1-7, C.sub.1-8, C.sub.2-3, C.sub.2-4, C.sub.2-5, C.sub.2-6, C.sub.3-4, C.sub.3-5, C.sub.3-6, C.sub.4-5, C.sub.4-6 and C.sub.5-6. For example, C.sub.1-6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. In some embodiments, alkyl groups have 1 to 12 carbon atoms. Alkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0059] As used herein, the term alkenyl refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C.sub.2, C.sub.2-3, C.sub.2-4, C.sub.2-5, C.sub.2-6, C.sub.2-7, C.sub.2-8, C.sub.2-9, C.sub.2-10, C.sub.3, C.sub.3-4, C.sub.3-5, C.sub.3-6, C.sub.4, C.sub.4-5, C.sub.4-6, C.sub.5, C.sub.5-6, and C.sub.6. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0060] As used herein, the term alkynyl refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C.sub.2, C.sub.2-3, C.sub.2-4, C.sub.2-5, C.sub.2-6, C.sub.2-7, C.sub.2-8, C.sub.2-9, C.sub.2-10, C.sub.3, C.sub.3-4, C.sub.3-5, C.sub.3-6, C.sub.4, C.sub.4-5, C.sub.4-6, C.sub.5, C.sub.5-6, and C.sub.6. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0061] As used herein, the term aryl refers to an aromatic carbon ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of carbon ring atoms, such as, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by a bond to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. Other aryl groups include benzyl, having a methylene linking group. Some aryl groups have from 6 to 12 ring members, such as phenyl, naphthyl or biphenyl. Other aryl groups have from 6 to 10 ring members, such as phenyl or naphthyl. Some other aryl groups have 6 ring members, such as phenyl. Aryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0062] As used herein, the term aralkyl denotes an arylalkyl group wherein the aryl and alkyl are as herein described. Preferred aralkyls contain a lower alkyl moiety. Exemplary aralkyl groups include benzyl, 2-phenethyl and naphthalenemethyl.
[0063] As used herein, the term cycloalkyl refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any number of carbons, such as C.sub.3-6, C.sub.4-6, C.sub.5-6, C.sub.3-8, C.sub.4-8, C.sub.5-8, and C.sub.6-8. In some embodiments, cycloalkyl groups include 3 to 10 carbon atoms in the ring assembly. In some embodiments, cycloalkyl groups contain 5 to 10 carbon atoms in the ring assembly. Saturated monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2] bicyclooctane, decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially unsaturated, having one or more double or triple bonds in the ring. Representative cycloalkyl groups that are partially unsaturated include, but are not limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbomene, and norbomadiene. Cycloalkyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0064] As used herein, the term heterocyclyl refers to a saturated ring system having from 3 to 12 ring members and from 1 to 4 heteroatoms selected from N, O and S. Additional heteroatoms including, but not limited to, B, Al, Si and P can also be present in a heterocycloalkyl group. The heteroatoms can be oxidized to form moieties such as, but not limited to, S(O) and S(O).sub.2. Heterocyclyl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 4 to 6, or 4 to 7 ring members. Any suitable number of heteroatoms can be included in the heterocyclyl groups, such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3 to 4. Examples of heterocyclyl groups include, but are not limited to, aziridine, azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane, thiirane, thietane, thiolane (tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine, dioxane, or dithiane. Heterocyclyl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0065] As used herein, the term heteroaryl refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5 of the ring atoms are a heteroatom such as N, O or S. Additional heteroatoms including, but not limited to, B, Al, Si and P can also be present in a heteroaryl group. The heteroatoms can be oxidized to form moieties such as, but not limited to, S(O) and S(O).sub.2. Heteroaryl groups can include any number of ring atoms, such as, 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitable number of heteroatoms can be included in the heteroaryl groups, such as 1, 2, 3, 4, or 5, or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8 ring members and from 1 to 4 heteroatoms, or from 5 to 8 ring members and from 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4 heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms. Examples of heteroaryl groups include, but are not limited to, pyrrole, pyridine, imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, and isoxazole. Heteroaryl groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0066] As used herein, the term alkoxy refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: i.e., alkyl-O. As for alkyl group, alkoxy groups can have any suitable number of carbon atoms, such as C.sub.1-6 or C.sub.1-4. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkoxy groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0067] As used herein, the term alkylthio refers to an alkyl group having a sulfur atom that connects the alkyl group to the point of attachment: i.e., alkyl-S. As for alkyl groups, alkylthio groups can have any suitable number of carbon atoms, such as C.sub.1-6 or C.sub.1-4. Alkylthio groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. Alkylthio groups can be optionally substituted with one or more moieties selected from halo, hydroxy, amino, alkylamino, alkoxy, haloalkyl, carboxy, amido, nitro, oxo, and cyano.
[0068] As used herein, the terms halo and halogen refer to fluorine, chlorine, bromine and iodine.
[0069] As used herein, the term haloalkyl refers to an alkyl moiety as defined above substituted with at least one halogen atom.
[0070] As used herein, the term alkylsilyl refers to a moiety SiR.sub.3, wherein at least one R group is alkyl and the other R groups are H or alkyl. The alkyl groups can be substituted with one more halogen atoms.
[0071] As used herein, the term acyl refers to a moiety C(O)R, wherein R is an alkyl group.
[0072] As used herein, the term oxo refers to an oxygen atom that is double-bonded to a compound (i.e., O).
[0073] As used herein, the term carboxy refers to a moiety C(O)OH. The carboxy moiety can be ionized to form the carboxylate anion.
[0074] As used herein, the term amino refers to a moiety NR.sub.3, wherein each R group is H or alkyl.
[0075] As used herein, the term amido refers to a moiety NRC(O)R or C(O)NR.sub.2, wherein each R group is H or alkyl.
III. Description of the Embodiments
[0076] The present invention provides a method by which trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine is prepared using synthetic strategies that are enabled by a biocatalytic step. The method includes incubating an olefinic substrate and a diazoester reagent or a diazoketone reagent with a cyclopropanation catalyst such as a heme enzyme to form a cyclopropane product. In some embodiments, the cyclopropane product is trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine. In other embodiments, the cyclopropane product is converted to trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine in one or more synthetic steps.
[0077] Accordingly, one aspect of the invention provides a method for producing a cyclopropanation product of Formula A:
##STR00010##
wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, C.sub.1-18 alkynyl, C.sub.1-18 alkoxy, C.sub.1-18 alkenyloxy, C.sub.1-18 alkynyloxy. The method includes combining an olefinic substrate, a carbene precursor, and a heme enzyme under conditions sufficient to form the product of Formula A. In some embodiments, R.sup.6 is C.sub.1-18 alkoxy and the carbene precursor is a diazoester. In some embodiments, R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, and C.sub.1-18 alkynyl and the carbene precursor is a diazoketone.
[0078] In a related aspect, the invention provides a reaction mixture for producing a cyclopropanation product of Formula A. The reaction mixture includes an olefinic substrate, a carbene precursor, and a heme enzyme as described above.
[0079] In some embodiments, the invention provides a method for preparing substituted phenylcyclopropylamine derivatives of Formula XLII:
##STR00011##
or a stereochemically isomeric form or a mixture of stereochemically isomeric forms thereof, or an acid addition salt thereof, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are, each independently, selected from hydrogen and a halogen atom, with the proviso that the benzene ring is substituted with at least one or more halogen atoms, wherein the halogen atom is F, Cl, Br or I, preferably, the halogen atom is F. The method includes:
a) reacting a halogen substituted benzaldehyde compound of Formula XLIII:
##STR00012##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are as defined in Formula XLII; with a methyltriphenyl phosphonium halide (Wittig reagent) of Formula XLIV:
##STR00013##
wherein X is a halogen, selected from the group consisting of Cl, Br and I;
in the presence of a first base in a first solvent to produce a substituted styrene compound of Formula XLV:
##STR00014##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are as defined above;
b) reacting the compound of Formula XLV with a diazoester compound of Formula XLVI:
##STR00015##
wherein R.sup.6a is an alkyl, cycloalkyl, aryl or aralkyl group; in the presence of a heme protein catalyst to produce a substituted cyclopropanecarboxylate compound of Formula XLVIIa:
##STR00016##
or a stereochemically isomeric form or a mixture of stereochemically isomeric forms thereof, wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 are as defined above;
c) hydrolyzing the ester compound of Formula XLVIIa with an acid or a second base in a third solvent to produce a substituted cyclopropanecarboxylic acid compound of Formula XLVIIIa:
##STR00017##
or a stereochemically isomeric form or a mixture of stereochemically isomeric forms thereof;
d) optionally, purifying the cyclopropanecarboxylic acid compound of Formula XLVIIIa by treating with a chiral amine in a fourth solvent to produce a pure chiral amine salt of the compound of Formula XLVIIIa;
e) optionally, acidifying the chiral amine salt of the compound of Formula XLVIIIa with an acid to produce a pure cyclopropanecarboxylic acid compound of Formula XLVIIIa;
f) reacting the cyclopropanecarboxylic acid compound of Formula XLVIIIa or a chiral amine salt thereof obtained in step-(c), (d) or (e) with an azide compound, with the proviso that the azide does not include sodium azide, in the presence a third base in a fifth solvent to produce an isocyanate intermediate, followed by subjecting to acidic hydrolysis with an acid in a sixth solvent and then basifying with a fourth base to produce the substituted phenylcyclopropylamine derivatives of Formula XLII or a stereochemically isomeric form or a mixture of stereochemically isomeric forms thereof, and optionally converting the compound of Formula XLII obtained into an acid addition salt thereof.
[0080] In some embodiments, the halogen atom X in the compound of Formula XLIV is Cl or Br, and more specifically, X is Br.
[0081] In some embodiments, in the compounds of Formulae XLII, XLIII, XLV, XLVIIa, and XLVIIIa, the R.sup.1, R.sup.4 and R.sup.5 are H, and the R.sup.2 and R.sup.3 are F.
[0082] The compounds can exist in different isomeric forms such as cis/trans isomers, enantiomers, or diastereomers. The method disclosed herein includes all such isomeric forms and mixtures thereof in all proportions.
[0083] In some embodiments, the group R.sup.6a in the compounds of Formulae XLVI and XLVIIa is selected from the group consisting of methyl, ethyl, isopropyl, tert-butyl, benzyl, L- or D-menthyl, and the like; and more specifically, R is ethyl.
[0084] In some embodiments, a specific substituted phenylcyclopropylamine derivative prepared by the methods described herein is trans-(1R, 2S)-2-(3,4-difluorophenyl)-cyclopropylamine of Formula IIa:
##STR00018##
[0085] In some embodiments, a specific substituted phenylcyclopropylamine derivative prepared by the methods described herein is trans-(1S,2R)-2-(3,4-difluorophenyl)-cyclopropylamine of Formula IIb:
##STR00019##
[0086] In some instances, the heme enzyme variant produces a plurality of cyclopropanation products having a plurality of the E cyclopropane products. Moreover, in some instances the heme enzymes described herein produce a plurality of the desired 1R, 2R cyclopropane carboxylate product of Formula VIIa with a % ee of 20% or greater.
##STR00020##
[0087] One skilled in the art will understand that suitably produced cyclopropane of Formula VII of variable enantioenrichment can be subjected to a hydrolase, lipase, or esterase enzyme that selectively hydrolyzes the ethyl ester of a single diastereomer of Formula VII to give exclusively or predominantly cyclopropane of Formula VIIIa or salt thereof, as shown in Scheme 7.
##STR00021##
[0088] One skilled in the art will understand that suitably produced cyclopropane of Formula VII of variable enantioenrichment can be subjected to a hydrolase, lipase, or esterase enzyme that selectively maintains the ethyl ester of a single diastereomer of VII to give exclusively or predominantly cyclopropane carboxylic acid ethyl ester of Formula VIIa, as shown in Scheme 8.
##STR00022##
[0089] A lipase isolated from Thermomyces lanuginosus (ALMAC lipase kit; AH-45) catalyzes the transformation shown in Scheme 8, and can provide excellent levels of enantioenrichment of the desired enantiomer as the ethyl ester of Formula VIIa.
[0090] The cyclopropane carboxylate ethyl ester of Formula VIIa can be selectively removed from the reaction milieu shown in Scheme 8 by selective extraction or distillation or chromatographic separation. Subsequent chemical hydrolysis or enzymatic hydrolysis of the ethyl ester of Formula VIIa will then yield the desired enantiopure or enantioenriched cyclopropane carboxylate of Formula VIIIa, which can then be converted to the desired compound of Formula IIa.
[0091] A. Heme Enzymes
[0092] In general, methods of the invention include the use of one or more heme enzymes that catalyze the conversion of an olefinic substrate to products containing one or more cyclopropane functional groups. In some embodiments, the present invention provides methods which use heme enzyme variants comprising at least one or more amino acid mutations therein that catalyze the formal transfer of carbene equivalents from a diazo reagent (e.g., a diazoester or a diazoketone) to an olefinic substrate, making cyclopropane products with high stereoselectivity. In certain embodiments, the heme enzyme variants of the present invention have the ability to catalyze cyclopropanation reactions efficiently, display increased total turnover numbers, and/or demonstrate highly regio- and/or enantioselective product formation compared to the corresponding wild-type enzymes.
[0093] The terms heme enzyme and heme protein are used herein to include any member of a group of proteins containing heme as a prosthetic group. Non-limiting examples of heme enzymes include globins, cytochromes, oxidoreductases, any other protein containing a heme as a prosthetic group, and combinations thereof. Heme-containing globins include, but are not limited to, hemoglobin, myoglobin, and combinations thereof. Heme-containing cytochromes include, but are not limited to, cytochrome P450, cytochrome b, cytochrome c1, cytochrome c, and combinations thereof. Heme-containing oxidoreductases include, but are not limited to, a catalase, an oxidase, an oxygenase, a haloperoxidase, a peroxidase, and combinations thereof.
[0094] Exemplary catalysts used in the cyclopropanation reactions include hemoproteins of the sort described in U.S. Pat. No. 8,993,262. In certain embodiments, the catalyst is comprised of a natural or engineered hemoprotein containing a histidine at the axial position of the heme coordination site. In particular embodiments, the heme enzyme comprises a histidine mutation at the axial position of the heme coordination site.
[0095] In certain instances, the heme enzymes are metal-substituted heme enzymes containing protoporphyrin IX or other porphyrin molecules containing metals other than iron, including, but not limited to, cobalt, rhodium, copper, ruthenium, and manganese, which are active cyclopropanation catalysts.
[0096] In certain embodiments, mutations can be introduced into a target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce heme enzyme variants (e.g., cytochrome P450 variants). Heme enzyme variants can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Cyclopropanation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.
[0097] The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the invention can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage PI-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
[0098] The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.
[0099] It is understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST)
[0100] Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE vectors (Qiagen), pB!uescript vectors (Stratagene), pNH vectors, lambdaZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors, pChlamy I vectors (Life Technologies, Carlsbad, Calif.), pGEMI (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Nonlimiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLACI vectors, pKLAC2 vectors (New England Biolabs), pQE vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.
[0101] The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.
[0102] Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli MIS, DH5a, DHIO, HBIOI, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces (e.g., K. lac tis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sj21 cells from Spodoptera frugiperda, Hi-Five cells, BT1-TN-5B 1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.
[0103] In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active cyclopropanation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo cyclopropanation reactions of the present invention.
[0104] One skilled in the art will appreciate that the hemoprotein catalysts described herein can be improved through the introduction of additional DNA mutations which alter the resulting amino acid sequence of the hemoprotein catalyst so as to generate a catalyst that is highly selective for the desired cyclopropane (for example, giving a % ee greater than 95%). In particular, there are many examples in the scientific literature that describe processes through which the enantioselectivity and activity of hemoprotein carbene-transfer catalysts can be optimized (Wang, Z. J. et al. Angew. Chem. Int. Ed. 2013, 52, 6928-6931; Heel T. et al. 2014, ChemBioChem, 15, 2556-2562; Coelho P. S. et al. Nat. Chem. Biol. 2013, 9, 485-487; Coelho P. S. et al. Science 2013, 339, 307-310). Specifically, one skilled in the art will know that through a process of random mutagenesis via error-prone PCR, or through a process of site-directed mutagenesis in which one or more codons are randomized sequentially or simultaneously, or through a process of gene synthesis in which random or directed mutations are introduced, many different mutants of the genes encoding the hemoprotein catalysts described herein can be generated.
[0105] One skilled in the art will understand that mutant genes encoding hemoprotein catalysts can be produced as hemoproteins in suitable hosts as described herein.
[0106] One skilled in the art will understand that suitably produced hemoprotein mutants can then be screened by various methods including but not limited to LC-MS, HPLC, GC, or SFC to determine whether one or several mutations introduced are beneficial for any desired parameter (% ee, % yield, specific activity, expression, solvent tolerance) that improves the hemoprotein-catalyzed synthesis of cyclopropanation products.
[0107] One skilled in the art will understand that hemoprotein mutants identified as improved in the synthesis of the cyclopropanation products can themselves be subjected to additional mutagenesis as described herein, resulting in progressive, cumulative improvements in one or more reaction parameters including but not limited to % ee, % yield, specific activity, expression, or solvent tolerance.
[0108] In some embodiments, the heme enzyme is a member of one of the enzyme classes set forth in Table 1. In other embodiments, the heme enzyme is a variant or homolog of a member of one of the enzyme classes set forth in Table 1. In yet other embodiments, the heme enzyme comprises or consists of the heme domain of a member of one of the enzyme classes set forth in Table 1 or a fragment thereof (e.g., a truncated heme domain) that is capable of carrying out the cyclopropanation reactions described herein.
TABLE-US-00001 TABLE 1 Heme enzymes identified by their enzyme classification number (EC number) and classification name. EC Number Name 1.1.2.3 L-lactate dehydrogenase 1.1.2.6 polyvinyl alcohol dehydrogenase (cytochrome) 1.1.2.7 methanol dehydrogenase (cytochrome c) 1.1.5.5 alcohol dehydrogenase (quinone) 1.1.5.6 formate dehydrogenase-N: 1.1.9.1 alcohol dehydrogenase (azurin): 1.1.99.3 gluconate 2-dehydrogenase (acceptor) 1.1.99.11 fructose 5-dehydrogenase 1.1.99.18 cellobiose dehydrogenase (acceptor) 1.1.99.20 alkan-1-ol dehydrogenase (acceptor) 1.2.1.70 glutamyl-tRNA reductase 1.2.3.7 indole-3-acetaldehyde oxidase 1.2.99.3 aldehyde dehydrogenase (pyrroloquinoline-quinone) 1.3.1.6 fumarate reductase (NADH): 1.3.5.1 succinate dehydrogenase (ubiquinone) 1.3.5.4 fumarate reductase (menaquinone) 1.3.99.1 succinate dehydrogenase 1.4.9.1 methylamine dehydrogenase (amicyanin) 1.4.9.2. aralkylamine dehydrogenase (azurin) 1.5.1.20 methylenetetrahydrofolate reductase [NAD(P)H] 1.5.99.6 spermidine dehydrogenase 1.6.3.1 NAD(P)H oxidase 1.7.1.1 nitrate reductase (NADH) 1.7.1.2 Nitrate reductase [NAD(P)H] 1.7.1.3 nitrate reductase (NADPH) 1.7.1.4 nitrite reductase [NAD(P)H] 1.7.1.14 nitric oxide reductase [NAD(P), nitrous oxide-forming] 1.7.2.1 nitrite reductase (NO-forming) 1.7.2.2 nitrite reductase (cytochrome; ammonia-forming) 1.7.2.3 trimethylamine-N-oxide reductase (cytochrome c) 1.7.2.5 nitric oxide reductase (cytochrome c) 1.7.2.6 hydroxylamine dehydrogenase 1.7.3.6 hydroxylamine oxidase (cytochrome) 1.7.5.1 nitrate reductase (quinone) 1.7.5.2 nitric oxide reductase (menaquinol) 1.7.6.1 nitrite dismutase 1.7.7.1 ferredoxin-nitrite reductase 1.7.7.2 ferredoxin-nitrate reductase 1.7.99.4 nitrate reductase 1.7.99.8 hydrazine oxidoreductase 1.8.1.2 sulfite reductase (NADPH) 1.8.2.1 sulfite dehydrogenase 1.8.2.2 thiosulfate dehydrogenase 1.8.2.3 sulfide-cytochrome-c reductase (flavocytochrome c) 1.8.2.4 dimethyl sulfide:cytochrome c2 reductase 1.8.3.1 sulfite oxidase 1.8.7.1 sulfite reductase (ferredoxin) 1.8.98.1 CoB-CoM heterodisulfide reductase 1.8.99.1 sulfite reductase 1.8.99.2 adenylyl-sulfate reductase 1.8.99.3 hydrogensulfite reductase 1.9.3.1 cytochrome-c oxidase 1.9.6.1 nitrate reductase (cytochrome) 1.10.2.2 ubiquinol-cytochrome-c reductase 1.10.3.1 catechol oxidase 1.10.3.B1 caldariellaquinol oxidase (H+-transporting) 1.10.3.3 L-ascorbate oxidase 1.10.3.9 photosystem II 1.10.3.10 ubiquinol oxidase (H+-transporting) 1.10.3.11 ubiquinol oxidase 1.10.3.12 menaquinol oxidase (H+-transporting) 1.10.9.1 plastoquinol-plastocyanin reductase 1.11.1.5 cytochrome-c peroxidase 1.11.1.6 catalase 1.11.1.7 peroxidase 1.11.1.B2 chloride peroxidase (vanadium-containing) 1.11.1.B7 bromide peroxidase (heme-containing) 1.11.1.8 iodide peroxidase 1.11.1.10 chloride peroxidase 1.11.1.11 L-ascorbate peroxidase 1.11.1.13 manganese peroxidase 1.11.1.14 lignin peroxidase 1.11.1.16 versatile peroxidase 1.11.1.19 dye decolorizing peroxidase 1.11.1.21 catalase-peroxidase 1.11.2.1 unspecific peroxygenase 1.11.2.2 myeloperoxidase 1.11.2.3 plant seed peroxygenase 1.11.2.4 fatty-acid peroxygenase 1.12.2.1 cytochrome-c3 hydrogenase 1.12.5.1 hydrogen:quinone oxidoreductase 1.12.99.6 hydrogenase (acceptor) 1.13.11.9 2,5-dihydroxypyridine 5,6-dioxygenase 1.13.11.11 tryptophan 2,3-dioxygenase 1.13.11.49 chlorite O2-lyase 1.13.11.50 acetylacetone-cleaving enzyme 1.13.11.52 indoleamine 2,3-dioxygenase 1.13.11.60 linoleate 8R-lipoxygenase 1.13.99.3 tryptophan 2-dioxygenase 1.14.11.9 flavanone 3-dioxygenase 1.14.12.17 nitric oxide dioxygenase 1.14.13.39 nitric-oxide synthase (NADPH dependent) 1.14.13.17 cholesterol 7alpha-monooxygenase 1.14.13.41 tyrosine N-monooxygenase 1.14.13.70 sterol 14alpha-demethylase 1.14.13.71 N-methylcoclaurine 3-monooxygenase 1.14.13.81 magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase 1.14.13.86 2-hydroxyisoflavanone synthase 1.14.13.98 cholesterol 24-hydroxylase 1.14.13.119 5-epiaristolochene 1,3-dihydroxylase 1.14.13.126 vitamin D3 24-hydroxylase 1.14.13.129 beta-carotene 3-hydroxylase 1.14.13.141 cholest-4-en-3-one 26-monooxygenase 1.14.13.142 3-ketosteroid 9alpha-monooxygenase 1.14.13.151 linalool 8-monooxygenase 1.14.13.156 1,8-cineole 2-endo-monooxygenase 1.14.13.159 vitamin D 25-hydroxylase 1.14.14.1 unspecific monooxygenase 1.14.15.1 camphor 5-monooxygenase 1.14.15.6 cholesterol monooxygenase (side-chain-cleaving) 1.14.15.8 steroid 15beta-monooxygenase 1.14.15.9 spheroidene monooxygenase 1.14.18.1 tyrosinase 1.14.19.1 stearoyl-CoA 9-desaturase 1.14.19.3 linoleoyl-CoA desaturase 1.14.21.7 biflaviolin synthase 1.14.99.1 prostaglandin-endoperoxide synthase 1.14.99.3 heme oxygenase 1.14.99.9 steroid 17alpha-monooxygenase 1.14.99.10 steroid 21-monooxygenase 1.14.99.15 4-methoxybenzoate monooxygenase (O-demethylating) 1.14.99.45 carotene epsilon-monooxygenase 1.16.5.1 ascorbate ferrireductase (transmembrane) 1.16.9.1 iron:rusticyanin reductase 1.17.1.4 xanthine dehydrogenase 1.17.2.2 lupanine 17-hydroxylase (cytochrome c) 1.17.99.1 4-methylphenol dehydrogenase (hydroxylating) 1.17.99.2 ethylbenzene hydroxylase 1.97.1.1 chlorate reductase 1.97.1.9 selenate reductase 2.7.7.65 diguanylate cyclase 2.7.13.3 histidine kinase 3.1.4.52 cyclic-guanylate-specific phosphodiesterase 4.2.1.B9 colneleic acid/etheroleic acid synthase 4.2.1.22 Cystathionine beta-synthase 4.2.1.92 hydroperoxide dehydratase 4.2.1.212 colneleate synthase 4.3.1.26 chromopyrrolate synthase 4.6.1.2 guanylate cyclase 4.99.1.3 sirohydrochlorin cobaltochelatase 4.99.1.5 aliphatic aldoxime dehydratase 4.99.1.7 phenylacetaldoxime dehydratase 5.3.99.3 prostaglandin-E synthase 5.3.99.4 prostaglandin-I synthase 5.3.99.5 Thromboxane-A synthase 5.4.4.5 9,12-octadecadienoate 8-hydroperoxide 8R-isomerase 5.4.4.6 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase 6.6.1.2 cobaltochelatase
[0109] In particular embodiments, the heme enzyme is a variant or a fragment thereof (e.g., a truncated variant containing the heme domain) comprising at least one mutation such as, e.g., a mutation at the axial position of the heme coordination site. In some instances, the mutation is a substitution of the native residue with Ala, Asp, Arg, Asn, Cys, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at the axial position. In certain instances, the mutation is a substitution of Cys with any other amino acid such as Ser at the axial position.
[0110] In certain embodiments, the in vitro methods for producing a cyclopropanation product comprise providing a heme enzyme, variant, or homolog thereof with a reducing agent such as NADPH or a dithionite salt (e.g., Na.sub.2S.sub.2O.sub.4). In certain other embodiments, the in vivo methods for producing a cyclopropanation product comprise providing whole cells such as E. coli cells expressing a heme enzyme, variant, or homolog thereof.
[0111] In some embodiments, the heme enzyme, variant, or homolog thereof is recombinantly expressed and optionally isolated and/or purified for carrying out the in vitro cyclopropanation reactions of the present invention. In other embodiments, the heme enzyme, variant, or homolog thereof is expressed in whole cells such as E. coli cells, and these cells are used for carrying out the in vivo cyclopropanation reactions of the present invention.
[0112] In certain embodiments, the heme enzyme, variant, or homolog thereof comprises or consists of the same number of amino acid residues as the wild-type enzyme (e.g., a full-length polypeptide). In some instances, the heme enzyme, variant, or homolog thereof comprises or consists of an amino acid sequence without the start methionine (e.g., P450 BM3 amino acid sequence set forth in SEQ ID NO:1). In other embodiments, the heme enzyme comprises or consists of a heme domain fused to a reductase domain. In yet other embodiments, the heme enzyme does not contain a reductase domain, e.g., the heme enzyme contains a heme domain only or a fragment thereof such as a truncated heme domain.
[0113] In some embodiments, the heme enzyme, variant, or homolog thereof has an enhanced cyclopropanation activity of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold compared to the corresponding wild-type heme enzyme.
[0114] In some embodiments, the heme enzyme, variant, or homolog thereof has a resting state reduction potential higher than that of NADH or NADPH.
[0115] In particular embodiments, the heme enzyme comprises a cytochrome P450 enzyme. Cytochrome P450 enzymes constitute a large superfamily of heme-thiolate proteins involved in the metabolism of a wide variety of both exogenous and endogenous compounds. Usually, they act as the terminal oxidase in multicomponent electron transfer chains, such as P450-containing monooxygenase systems. Members of the cytochrome P450 enzyme family catalyze myriad oxidative transformations, including, e.g., hydroxylation, epoxidation, oxidative ring coupling, heteroatom release, and heteroatom oxygenation (E. M. Isin et al., Biochim. Biophys. Acta 1770, 314 (2007)). The active site of these enzymes contains an Fe.sup.III-protoporphyrin IX cofactor (heme) ligated proximally by a conserved cysteine thiolate (M. T. Green, Current Opinion in Chemical Biology 13, 84 (2009)). The remaining axial iron coordination site is occupied by a water molecule in the resting enzyme, but during native catalysis, this site is capable of binding molecular oxygen. In the presence of an electron source, typically provided by NADH or NADPH from an adjacent fused reductase domain or an accessory cytochrome P450 reductase enzyme, the heme center of cytochrome P450 activates molecular oxygen, generating a high valent iron(IV)-oxo porphyrin cation radical species intermediate (Compound I,
[0116] In certain embodiments the heme enzyme is a cytochrome P450 enzyme or a variant thereof. In a particular embodiment the cytochrome P450 enzyme is a P450 BM3 (also known as CYP102A1) enzyme or a variant thereof. In a further embodiment, the P450 BM3 enzyme comprises an axial ligand mutation C400H with or without an additional mutation at T268 to any other amino acid. In a further embodiment the P450 BM3 enzyme comprises a double mutant having an axial ligand mutation C400H and a further mutation of T268A. In certain embodiments, the CYP102A1 variants comprise the mutations T268A, C400H, L437W, V78M and L181V. These are termed BM3 Hstar and variants thereof, which are described in U.S. Pat. Appl. Pub. No. 2016/0032330 which is incorporated herein by reference in its entirety (see also, Angew. Chem. Int. Ed. 2014, 53, 6810-6813).
[0117] In certain embodiments the heme enzyme is a cytochrome P450 enzyme or a variant thereof. In a particular embodiment the cytochrome P450 enzyme is a variant of the P450 enzyme CYP119 (Swiss-Prot: Q55080.1) from Sulfolobus acidocaldarius DSM 639 that contains a mutation at the axial heme ligand (residue C317) to any other amino acid residue that is among the naturally occurring twenty amino acids. In a further embodiment, the CYP119 enzyme consists of a single mutation of the position T213A to any other amino acid. In a further embodiment, the CYP119 enzyme consists of a axial ligand mutation C317 to any other amino acid along with a mutation of the position T213 to any other amino acid.
[0118] In certain embodiments the heme enzyme is a variant of the P450 enzyme CYP119 that contains mutations at either or both position C317 and H315. Each of these positions may be mutated to any other amino acid.
[0119] In some embodiments, the heme enzyme is a cytochrome P450 holoenzyme or a variant thereof. In certain embodiments, the heme enzyme is a cytochrome P450 heme domain or a variant thereof.
[0120] One skilled in the art will appreciate that the cytochrome P450 enzyme superfamily has been compiled in various databases, including, but not limited to, the P450 homepage (available at http://dmelson.uthsc.edu/CytochromeP450.html; see also, D. R. Nelson, Hum. Genomics 4, 59 (2009)), the cytochrome P450 enzyme engineering database (available at http://www.cyped.uni-stuttgart.de/cgi-bin/CYPED5/index.pl; see also, D. Sirim et al., BMC Biochem 10, 27 (2009)), and the SuperCyp database (available at http://bioinformatics.charite.de/supercyp/; see also, S. Preissner et al., Nucleic Acids Res. 38, D237 (2010)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.
[0121] In certain embodiments, the cytochrome P450 enzymes used in the methods of present the invention are members of one of the classes shown in Table 2 (see, http://www.icgeb.org/p450srv/P450enzymes.html, the disclosure of which is incorporated herein by reference in its entirety for all purposes).
TABLE-US-00002 TABLE 2 Cytochrome P450 enzymes classified by their EC number, recommended name, and family/gene name. EC Recommended name Family/gene 1.3.3.9 secologanin synthase CYP72A1 1.14.13.11 trans-cinnamate 4-monooxygenase CYP73 1.14.13.12 benzoate 4-monooxygenase CYP53 1.14.13.13 calcidiol 1-monooxygenase CYP27 1.14.13.15 cholestanetriol 26-monooxygenase CYP27 1.14.13.17 cholesterol 7-monooxygenase CYP7 1.14.13.21 flavonoid 3-monooxygenase CYP75 1.14.13.28 3,9-dihydroxypterocarpan 6a-monooxygenase CYP93A1 1.14.13.30 leukotriene-B.sub.4 20-monooxygenase CYP4F 1.14.13.37 methyltetrahydroprotoberberine CYP93A1 14-monooxygenase 1.14.13.41 tyrosine N-monooxygenase CYP79 1.14.13.42 hydroxyphenylacetonitrile 2-monooxygenase 1.14.13.47 ()-limonene 3-monooxygenase 1.14.13.48 ()-limonene 6-monooxygenase 1.14.13.49 ()-limonene 7-monooxygenase 1.14.13.52 isoflavone 3-hydroxylase 1.14.13.53 isoflavone 2-hydroxylase 1.14.13.55 protopine 6-monooxygenase 1.14.13.56 dihydrosanguinarine 10-monooxygenase 1.14.13.57 dihydrochelirubine 12-monooxygenase 1.14.13.60 27-hydroxycholesterol 7-monooxygenase 1.14.13.70 sterol 14-demethylase CYP51 1.14.13.71 N-methylcoclaurine 3-monooxygenase CYP80B1 1.14.13.73 tabersonine 16-hydroxylase CYP71D12 1.14.13.74 7-deoxyloganin 7-hydroxylase 1.14.13.75 vinorine hydroxylase 1.14.13.76 taxane 10-hydroxylase CYP725A1 1.14.13.77 taxane 13-hydroxylase CYP725A2 1.14.13.78 ent-kaurene oxidase CYP701 1.14.13.79 ent-kaurenoic acid oxidase CYP88A 1.14.14.1 unspecific monooxygenase multiple 1.14.15.1 camphor 5-monooxygenase CYP101 1.14.15.3 alkane 1-monooxygenase CYP4A 1.14.15.4 steroid 11-monooxygenase CYP11B 1.14.15.5 corticosterone 18-monooxygenase CYP11B 1.14.15.6 cholesterol monooxygenase CYP11A (side-chain-cleaving) 1.14.21.1 (S)-stylopine synthase 1.14.21.2 (S)-cheilanthifoline synthase 1.14.21.3 berbamunine synthase CYP80 1.14.21.4 salutaridine synthase 1.14.21.5 (S)-canadine synthase 1.14.99.9 steroid 17-monooxygenase CYP17 1.14.99.10 steroid 21-monooxygenase CYP21 1.14.99.22 ecdysone 20-monooxygenase 1.14.99.28 linalool 8-monooxygenase CYP111 4.2.1.92 hydroperoxide dehydratase CYP74 5.3.99.4 prostaglandin-I synthase CYP8 5.3.99.5 thromboxane-A synthase CYP5
[0122] Table 3 below lists additional cyctochrome P450 enzymes that are suitable for use in the cyclopropanation reactions of the present invention. The accession numbers in Table 3 are incorporated herein by reference in their entirety for all purposes. The cytochrome P450 gene and/or protein sequences disclosed in the following patent documents are hereby incorporated by reference in their entirety for all purposes: WO 2013/076258; CN 103160521; CN 103223219; KR 2013081394; JP 5222410; WO 2013/073775; WO 2013/054890; WO 2013/048898; WO 2013/031975; WO 2013/064411; U.S. Pat. No. 8,361,769; WO 2012/150326, CN 102747053; CN 102747052; JP 2012170409; WO 2013/115484; CN 103223219; KR 2013081394; CN 103194461; JP 5222410; WO 2013/086499; WO 2013/076258; WO 2013/073775; WO 2013/064411; WO 2013/054890; WO 2013/031975; U.S. Pat. No. 8,361,769; WO 2012/156976; WO 2012/150326; CN 102747053; CN 102747052; US 20120258938; JP 2012170409; CN 102399796; JP 2012055274; WO 2012/029914; WO 2012/028709; WO 2011/154523; JP 2011234631; WO 2011/121456; EP 2366782; WO 2011/105241; CN 102154234; WO 2011/093185; WO 2011/093187; WO 2011/093186; DE 102010000168; CN 102115757; CN 102093984; CN 102080069; JP 2011103864; WO 2011/042143; WO 2011/038313; JP 2011055721; WO 2011/025203; JP 2011024534; WO 2011/008231; WO 2011/008232; WO 2011/005786; IN 2009DE01216; DE 102009025996; WO 2010/134096; JP 2010233523; JP 2010220609; WO 2010/095721; WO 2010/064764; US 20100136595; JP 2010051174; WO 2010/024437; WO 2010/011882; WO 2009/108388; US 20090209010; US 20090124515; WO 2009/041470; KR 2009028942; WO 2009/039487; WO 2009/020231; JP 2009005687; CN 101333520; CN 101333521; US 20080248545; JP 2008237110; CN 101275141; WO 2008/118545; WO 2008/115844; CN 101255408; CN 101250506; CN 101250505; WO 2008/098198; WO 2008/096695; WO 2008/071673; WO 2008/073498; WO 2008/065370; WO 2008/067070; JP 2008127301; JP 2008054644; KR 794395; EP 1881066; WO 2007/147827; CN 101078014; JP 2007300852; WO 2007/048235; WO 2007/044688; WO 2007/032540; CN 1900286; CN 1900285; JP 2006340611; WO 2006/126723; KR 2006029792; KR 2006029795; WO 2006/105082; WO 2006/076094; US 2006/0156430; WO 2006/065126; JP 2006129836; CN 1746293; WO 2006/029398; JP 2006034215; JP 2006034214; WO 2006/009334; WO 2005/111216; WO 2005/080572; US 2005/0150002; WO 2005/061699; WO 2005/052152; WO 2005/038033; WO 2005/038018; WO 2005/030944; JP 2005065618; WO 2005/017106; WO 2005/017105; US 20050037411; WO 2005/010166; JP 2005021106; JP 2005021104; JP 2005021105; WO 2004/113527; CN 1472323; JP 2004261121; WO 2004/013339; WO 2004/011648; DE 10234126; WO 2004/003190; WO 2003/087381; WO 2003/078577; US 20030170627; US 20030166176; US 20030150025; WO 2003/057830; WO 2003/052050; CN 1358756; US 20030092658; US 20030078404; US 20030066103; WO 2003/014341; US 20030022334; WO 2003/008563; EP 1270722; US 20020187538; WO 2002/092801; WO 2002/088341; US 20020160950; WO 2002/083868; US 20020142379; WO 2002/072758; WO 2002/064765; US 20020076777; US 20020076774; US 20020076774; WO 2002/046386; WO 2002/044213; US 20020061566; CN 1315335; WO 2002/034922; WO 2002/033057; WO 2002/029018; WO 2002/018558; JP 2002058490; US 20020022254; WO 2002/008269; WO 2001/098461; WO 2001/081585; WO 2001/051622; WO 2001/034780; CN 1271005; WO 2001/011071; WO 2001/007630; WO 2001/007574; WO 2000/078973; U.S. Pat. No. 6,130,077; JP 2000152788; WO 2000/031273; WO 2000/020566; WO 2000/000585; DE 19826821; JP 11235174; U.S. Pat. No. 5,939,318; WO 99/19493; WO 99/18224; U.S. Pat. No. 5,886,157; WO 99/08812; U.S. Pat. No. 5,869,283; JP 10262665; WO 98/40470; EP 776974; DE 19507546; GB 2294692; U.S. Pat. No. 5,516,674; JP 07147975; WO 94/29434; JP 06205685; JP 05292959; JP 04144680; DD 298820; EP 477961; SU 1693043; JP 01047375; EP 281245; JP 62104583; JP 63044888; JP 62236485; JP 62104582; and JP 62019084.
TABLE-US-00003 TABLE 3 Additional cytochrome P450 enzymes for use in the present invention. SEQ Species Cyp No. Accession No. ID NO Bacillus megaterium 102A1 AAA87602 1 Bacillus megaterium 102A1 ADA57069 2 Bacillus megaterium 102A1 ADA57068 3 Bacillus megaterium 102A1 ADA57062 4 Bacillus megaterium 102A1 ADA57061 5 Bacillus megaterium 102A1 ADA57059 6 Bacillus megaterium 102A1 ADA57058 7 Bacillus megaterium 102A1 ADA57055 8 Bacillus megaterium 102A1 ACZ37122 9 Bacillus megaterium 102A1 ADA57057 10 Bacillus megaterium 102A1 ADA57056 11 Mycobacterium sp. HXN-1500 153A6 CAH04396 12 Tetrahymena thermophile 5013C2 ABY59989 13 Nonomuraea dietziae AGE14547.1 14 Homo sapiens 2R1 NP_078790 15 Macca mulatta 2R1 NP_001180887.1 16 Canis familiaris 2R1 XP_854533 17 Mus musculus 2R1 AAI08963 18 Bacillus halodurans C-125 152A6 NP_242623 19 Streptomyces parvus aryC AFM80022 20 Pseudomonas putida 101A1 P00183 21 Homo sapiens 2D7 AAO49806 22 Rattus norvegicus C27 AAB02287 23 Oryctolagus cuniculus 2B4 AAA65840 24 Bacillus subtilis 102A2 O08394 25 Bacillus subtilis 102A3 O08336 26 B. megaterium DSM 32 102A1 P14779 27 B. cereus ATCC14579 102A5 AAP10153 28 B. licheniformis ATTC1458 102A7 YP 079990 29 B. thuringiensis serovar X YP 037304 30 konkukian str.97-27 R. metallidurans CH34 102E1 YP 585608 31 A. fumigatus Af293 505X EAL92660 32 A. nidulans FGSC A4 505A8 EAA58234 33 A. oryzae ATCC42149 505A3 Q2U4F1 34 A. oryzae ATCC42149 X Q2UNA2 35 F. oxysporum 505A1 Q9Y8G7 36 G. moniliformis X AAG27132 37 G. zeae PH1 505A7 EAA67736 38 G. zeae PH1 505C2 EAA77183 39 M. grisea 70-15 syn 505A5 XP 365223 40 N. crassa OR74 A 505A2 XP 961848 41 Oryza sativa* 97A Oryza sativa* 97B Oryza sativa 97C ABB47954 42 The start methionine (M) may be present or absent from these sequences. *See, M. Z. Lv et al., Plant Cell Physiol., 53(6): 987-1002 (2012).
[0123] In certain embodiments, the present invention provides amino acid substitutions that efficiently remove monooxygenation chemistry from cytochrome P450 enzymes. This system permits selective enzyme-driven cyclopropanation chemistry without competing side reactions mediated by native P450 catalysis. The invention also provides P450-mediated catalysis that is competent for cyclopropanation chemistry but not able to carry out traditional P450-mediated monooxygenation reactions as orthogonal P450 catalysis and respective enzyme variants as orthogonal P450s. In some instances, orthogonal P450 variants comprise a single amino acid mutation at the axial position of the heme coordination site (e.g., a C400S mutation in the P450 BM3 enzyme) that alters the proximal heme coordination environment. Accordingly, the present invention also provides P450 variants that contain an axial heme mutation in combination with one or more additional mutations described herein to provide orthogonal P450 variants that show enriched diastereoselective and/or enantioselective product distributions. The present invention further provides a compatible reducing agent for orthogonal P450 cyclopropanation catalysis that includes, but is not limited to, NAD(P)H or sodium dithionite.
[0124] In particular embodiments, the cytochrome P450 enzyme is one of the P450 enzymes or enzyme classes set forth in Table 2 or 3. In some embodiments, the cytochrome P450 enzyme is a variant or homolog of one of the P450 enzymes or enzyme classes set forth in Table 2 or 3. In preferred embodiments, the P450 enzyme variant comprises a mutation at the conserved cysteine (Cys or C) residue of the corresponding wild-type sequence that serves as the heme axial ligand to which the iron in protoporphyrin IX is attached. As non-limiting examples, axial mutants of any of the P450 enzymes set forth in Table 2 or 3 can comprise a mutation at the axial position (AxX) of the heme coordination site, wherein X is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
[0125] In certain embodiments, the conserved cysteine residue in a cytochrome P450 enzyme of interest that serves as the heme axial ligand and is attached to the iron in protoporphyrin IX can be identified by locating the segment of the DNA sequence in the corresponding cytochrome P450 gene which encodes the conserved cysteine residue. In some instances, this DNA segment is identified through detailed mutagenesis studies in a conserved region of the protein (see, e.g., Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances, the conserved cysteine is identified through crystallographic study (see, e.g., Poulos et al., J. Mol. Biol 195:687-700, 1987).
[0126] In situations where detailed mutagenesis studies and crystallographic data are not available for a cytochrome P450 enzyme of interest, the axial ligand may be identified through phylogenetic study. Due to the similarities in amino acid sequence between P450 enzymes, standard protein alignment algorithms may show a phylogenetic similarity between a P450 enzyme for which crystallographic or mutagenesis data exist and a new P450 enzyme for which such data do not exist. Thus, the polypeptide sequences of the present invention for which the heme axial ligand is known can be used as a query sequence to perform a search against a specific new cytochrome P450 enzyme of interest or a database comprising cytochrome P450 sequences to identify the heme axial ligand. Such analyses can be performed using the BLAST programs (see, e.g., Altschul et al., J Mol Biol. 215(3):403-10(1990)). Software for performing BLAST analyses publicly available through the National Center for Biotechnology Information (http://ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences.
[0127] Exemplary parameters for performing amino acid sequence alignments to identify the heme axial ligand in a P450 enzyme of interest using the BLASTP algorithm include E value=10, word size=3, Matrix=Blosum62, Gap opening=11, gap extension=1, and conditional compositional score matrix adjustment. Those skilled in the art will know what modifications can be made to the above parameters, e.g., to either increase or decrease the stringency of the comparison and/or to determine the relatedness of two or more sequences.
[0128] In preferred embodiments, the cytochrome P450 enzyme is a cytochrome P450 BM3 enzyme or a variant, homolog, or fragment thereof. The bacterial cytochrome P450 BM3 from Bacillus megaterium is a water soluble, long-chain fatty acid monooxygenase. The native P450 BM3 protein is comprised of a single polypeptide chain of 1048 amino acids and can be divided into 2 functional subdomains (see, L. O. Narhi et al., J. Biol. Chem. 261, 7160 (1986)). An N-terminal domain, amino acid residues 1-472, contains the heme-bound active site and is the location for monooxygenation catalysis. The remaining C-terminal amino acids encompass a reductase domain that provides the necessary electron equivalents from NADPH to reduce the heme cofactor and drive catalysis. The presence of a fused reductase domain in P450 BM3 creates a self-sufficient monooxygenase, obviating the need for exogenous accessory proteins for oxygen activation (see, id.). It has been shown that the N-terminal heme domain can be isolated as an individual, well-folded, soluble protein that retains activity in the presence of hydrogen peroxide as a terminal oxidant under appropriate conditions (P. C. Cirino et al., Angew. Chem., Int. Ed. 42, 3299 (2003)).
[0129] In certain instances, the cytochrome P450 BM3 enzyme comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1. In certain other instances, the cytochrome P450 BM3 enzyme is a natural variant thereof as described, e.g., in J. Y. Kang et al., AMB Express 1:1 (2011), wherein the natural variants are divergent in amino acid sequence from the wild-type cytochrome P450 BM3 enzyme sequence (SEQ ID NO:1) by up to about 5% (e.g., SEQ ID NOS:2-11).
[0130] In particular embodiments, the P450 BM3 enzyme variant comprises or consists of the heme domain of the wild-type P450 BM3 enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO:1) and optionally at least one mutation as described herein. In other embodiments, the P450 BM3 enzyme variant comprises or consists of a fragment of the heme domain of the wild-type P450 BM3 enzyme sequence (SEQ ID NO: 1), wherein the fragment is capable of carrying out the cyclopropanation reactions of the present invention. In some instances, the fragment includes the heme axial ligand and at least one, two, three, four, or five of the active site residues.
[0131] In certain embodiments, the P450 BM3 enzyme variant comprises a mutation at the axial position (AxX) of the heme coordination site, wherein X is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. The conserved cysteine (Cys or C) residue in the wild-type P450 BM3 enzyme is located at position 400 in SEQ ID NO:1. As used herein, the terms AxX and C400X refer to the presence of an amino acid substitution X located at the axial position (i.e., residue 400) of the wild-type P450 BM3 enzyme (i.e., SEQ ID NO:1). In some instances, X is Ser (S). In other instances, X is Ala (A), Asp (D), His (H), Lys (K), Asn (N), Met (M), Thr (T), or Tyr (Y). In some embodiments, the P450 BM3 enzyme variant comprises or consists of the heme domain of the wild-type P450 BM3 enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO: 1) or a fragment thereof and an AxX mutation (i.e., WT-AxX heme).
[0132] In other embodiments, the P450 BM3 enzyme variant comprises at least one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all thirteen) of the following amino acid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K. In certain instances, the P450 BM3 enzyme variant comprises a T268A mutation alone or in combination with one or more additional mutations such as a C400X mutation (e.g., C400S) in SEQ ID NO: 1. In other instances, the P450 BM3 enzyme variant comprises all thirteen of these amino acid substitutions (i.e., V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K; BM3-CIS) in combination with a C400X mutation (e.g., C400S) in SEQ ID NO:1. In some instances, the P450 BM3 enzyme variant comprises or consists of the heme domain of the BM3-CIS enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO: 1 comprising all thirteen of these amino acid substitutions) or a fragment thereof and an AxX mutation (i.e., BM3-CIS-AxX heme).
[0133] In some embodiments, the P450 BM3 enzyme variant further comprises at least one or more (e.g., at least two, or all three) of the following amino acid substitutions in SEQ ID NO:1: I263A, A328G, and a T438 mutation. In certain instances, the T438 mutation is T438A, T438S, or T438P. In some instances, the P450 BM3 enzyme variant comprises a T438 mutation such as T438A, T438S, or T438P alone or in combination with one or more additional mutations such as a C400X mutation (e.g., C400S) in SEQ ID NO:1 or a heme domain or fragment thereof. In other instances, the P450 BM3 enzyme variant comprises a T438 mutation such as T438A, T438S, or T438P in a BM3-CIS backbone alone or in combination with a C400X mutation (e.g., C400S) in SEQ ID NO:1 (i.e., BM3-CIS-T438S-AxX). In yet other instances, the P450 BM3 enzyme variant comprises or consists of the heme domain of the BM3-CIS enzyme sequence or a fragment thereof in combination with a T438 mutation and an AxX mutation (e.g., BM3-CIS-T438S-AxX heme).
[0134] In other embodiments, the P450 BM3 enzyme variant further comprises from one to five (e.g., one, two, three, four, or five) active site alanine substitutions in the active site of SEQ ID NO: 1. In certain instances, the active site alanine substitutions are selected from the group consisting of L75A, M177A, L181A, I263A, L437A, and a combination thereof.
[0135] In further embodiments, the P450 BM3 enzyme variant comprises at least one or more (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following amino acid substitutions in SEQ ID NO:1: R47C, L52I, I58V, L75R, F81 (e.g., F81L, F81W), A82 (e.g., A82S, A82F, A82G, A82T, etc.), F87A, K94I, I94K, H100R, S106R, F107L, A135S, F162I, A197V, F205C, N239H, R255S, S274T, L324I, A328V, V340M, and K434E. In particular embodiments, the P450 BM3 enzyme variant comprises any one or a plurality of these mutations alone or in combination with one or more additional mutations such as those described above, e.g., an AxX mutation and/or at least one or more mutations including V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K.
[0136] Table 4 below provides non-limiting examples of cytochrome P450 BM3 variants of the present invention. Each P450 BM3 variant comprises one or more of the listed mutations (Variant Nos. 1-31), wherein a + indicates the presence of that particular mutation(s) in the variant. Any of the variants listed in Table 4 can further comprise an I263A and/or an A328G mutation and/or at least one, two, three, four, or five of the following alanine substitutions, in any combination, in the P450 BM3 enzyme active site: L75A, M177A, L181A, I263A, and L437A. In particular embodiments, the P450 BM3 variant comprises or consists of the heme domain of any one of Variant Nos. 1-31 listed in Table 4 or a fragment thereof, wherein the fragment is capable of carrying out the cyclopropanation reactions of the present invention.
TABLE-US-00004 TABLE 4 Exemplary cytochrome P450 BM3 enzyme variants of the present invention. P450.sub.BM3 variant Mutation 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C400X + + + + + + T268A + + + + + + F87V + + + + + + 9-10A-TS + + + + + T438Z + + + + + P450.sub.BM3 variant Mutation 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 C400X + + + + + + + + + + T268A + + + + + + + + + + F87V + + + + + + + + + + 9-10A-TS + + + + + + + + + + + T438Z + + + + + + + + + + + Mutations relative to the wild-type P450.sub.BM3 amino acid sequence (SEQ ID NO: 1); X is selected from Ala, Asp, Arg, Asn, Glu, Gln, Gly, His, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; Z is selected from Ala, Ser, and Pro; 9-10A-TS includes the following amino acid substitutions in SEQ ID NO: 1: V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, and E442K.
[0137] One skilled in the art will understand that any of the mutations listed in Table 4 can be introduced into any cytochrome P450 enzyme of interest by locating the segment of the DNA sequence in the corresponding cytochrome P450 gene which encodes the conserved amino acid residue as described above for identifying the conserved cysteine residue in a cytochrome P450 enzyme of interest that serves as the heme axial ligand. In certain instances, this DNA segment is identified through detailed mutagenesis studies in a conserved region of the protein (see, e.g., Shimizu et al., Biochemistry 27, 4138-4141, 1988). In other instances, the conserved amino acid residue is identified through crystallographic study (see, e.g., Poulos et al., J. Mol. Biol 195:687-700, 1987). In yet other instances, protein sequence alignment algorithms can be used to identify the conserved amino acid residue. As non-limiting examples, BLAST alignment can be used with the P450 BM3 amino acid sequence as the query sequence to identify the heme axial ligand site and/or the equivalent T268 residue in other cytochrome P450 enzymes.
[0138] Table 5A below provides non-limiting examples of preferred cytochrome P450 BM3 variants of the present invention. Table 5B below provides non-limiting examples of preferred chimeric cytochrome P450 enzymes of the present invention.
TABLE-US-00005 TABLE 5A Exemplary cytochrome P450 BM3 enzyme variants for use in the invention. P450.sub.BM3 variants Mutations compared to wild-type P450.sub.BM3 (SEQ ID NO: 1) P450.sub.BM3-T268A T268A P450.sub.BM3-T268A-C400H T268A + C400H P411.sub.BM3 (ABC) C400S P411.sub.BM3-T268A T268A + C400S P450.sub.BM3-T268A-F87V T268A + F87V 9-10A TS V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K 9-10A-TS-F87V 9-10A TS + F87V H2A10 9-10A TS + F87V, L75A, L181A, T268A H2A10-C400S 9-10A TS + F87V, L75A, L181A, T268A, C400S H2A10-C400H 9-10A TS + F87V, L75A, L181A, T268A, C400H H2A10-C400M 9-10A TS + F87V, L75A, L181A, T268A, C400M H2-5-F10 9-10A TS + F87V, L75A, I263A, T268A, L437A H2-5-F10-C400S 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400S H2-5-F10-C400H 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400H H2-5-F10-C400M 9-10A TS + F87V, L75A, I263A, T268A, L437A, C400M H2-4-D4 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A H2-4-D4-C400S 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A, C400S H2-4-D4-C400H 9-10A TS + F87V, L75A, M177A, L181A, T268A, L437A, C400H H2-2-A1 9-10A TS + F87A, L75A, L181A, L437A H2-2-A1-C400S 9-10A TS + F87A, L75A, L181A, L437A, C400S H2-2-A1-C400H 9-10A TS + F87A, L75A, L181A, L437A, C400H H2-8-C7 9-10A TS + L75A, F87V, L181A H2-5-F10-A75L 9-10A TS + F87V-I263A-T268A-L437A CH F8 R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, F81W, A82S, F87A, A197V BM3-CIS (P450.sub.BM3-CIS; C3C) 9-10A TS + F87V, T268A BM3-CIS-I263A BM3-CIS + I263A BM3-CIS-A328G BM3-CIS + A328G BM3-CIS-T438S BM3-CIS + T438S BM3-CIS-C400S (P411.sub.BM3-CIS; BM3-CIS + C400S ABC-CIS) BM3-CIS-C400D (BM3-CIS-AxD) BM3-CIS + C400D BM3-CIS-C400Y (BM3-CIS-AxY) BM3-CIS + C400Y BM3-CIS-C400K (BM3-CIS-AxK) BM3-CIS + C400K BM3-CIS-C400H (BM3-CIS-AxH) BM3-CIS + C400H BM3-CIS-C400M (BM3-CIS-AxM) BM3-CIS + C400M WT-AxA (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400A WT-AxD (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400D WT-AxH (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400H WT-AxK (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400K WT-AxM (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400M WT-AxN (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400N WT-AxY (heme) WT heme domain (amino acids 1-463 of SEQ ID NO: 1) + C400Y BM3-CIS-T438S-AxA BM3-CIS-T438S + C400A BM3-CIS-T438S-AxD BM3-CIS-T438S + C400D BM3-CIS-T438S-AxM BM3-CIS-T438S + C400M BM3-CIS-T438S-AxY BM3-CIS-T438S + C400Y BM3-CIS-T438S-AxT BM3-CIS-T438S + C400T 7-11D R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, A82F, A328V 7-11D-C400S R47C, V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, A82F, A328V, C400S 12-10C R47C, V78A, A82G, F87V, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, A328V, L353V 22A3 L52I, I58V, L75R, F87A, H100R, S106R, F107L, A135S, F162I, A184V, N239H, S274T, L324I, V340M, I366V, K434E Man1 V78A, K94I, P142S, T175I, A184V, F205C, S226R, H236Q, E252G, R255S, A290V, L353V, F81L, A82T, F87A, I94K
TABLE-US-00006 TABLE 5B Exemplary chimeric cytochrome P450 enzymes for use the invention. Chimeric P450s Heme domain block sequence SEQ ID NO C2G9 22223132 43 X7 22312333 44 X7-12 12112333 45 C2E6 11113311 46 X7-9 32312333 47 C2B12 32313233 48 TSP234 22313333 49
[0139] In particular embodiments, cytochrome P450 BM3 variants with at least one or more amino acid mutations such as, e.g., C400X (AxX), BM3-CIS, T438, and/or T268A amino acid substitutions catalyze cyclopropanation reactions efficiently, displaying increased total turnover numbers and demonstrating highly regio- and/or enantioselective product formation compared to the wild-type enzyme.
[0140] As a non-limiting example, certain cytochrome P450 BM3 variants of the present invention are cis-selective catalysts that demonstrate diastereomeric ratios at least comparable to wild-type P450 BM3, e.g., at least 37:63 cis:trans, at least 50:50 cis:trans, at least 60:40 cis:trans, or at least 95:5 cis:trans. Particular mutations for improving cis-selective catalysis include at least one mutation comprising T268A, C400X, and T438S, but preferably one, two, or all three of these mutations in combination with additional mutations comprising V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K, and F87V derived from P450 BM3 variant 9-10A-TS. These mutations are isolated to the heme domain of P450 BM3 and are located in various regions of the heme domain structure including the active site and periphery.
[0141] As another non-limiting example, certain cytochrome P450 BM3 variants of the present invention are trans-selective catalysts that demonstrate diastereomeric ratios at least comparable to wild-type P450 BM3, e.g., at least 37:63 cis:trans, at least 20:80 cis:trans, or at least 1:99 cis:trans. Particular mutations for improving trans-selective catalysis include at least one mutation comprising including T268A and C400X, but preferably one or both of these mutations in the background of wild-type P450 BM3. In certain embodiments, trans-preferential mutations in combination with additional mutations such as V78A, P142S, T175I, A184V, S226R, H236Q, E252G, A290V, L353V, I366V, E442K, and F87V (from 9-10-A-TS) are also tolerated when in the presence of additional mutations including, but not limited to, I263A, L437A, L181A and/or L75A. These mutations are isolated to the heme domain of P450 BM3 and are located in various regions of the heme domain structure including the active site and periphery.
[0142] In certain embodiments, the present invention also provides P450 variants that catalyze enantioselective cyclopropanation with enantiomeric excess values of at least 30% (comparable with wild-type P450 BM3), but more preferably at least 80%, and even more preferably at least >95% for preferred product diastereomers.
[0143] In other aspects, the present invention provides chimeric heme enzymes such as, e.g., chimeric P450 proteins comprised of recombined sequences from P450 BM3 and at least one, two, or more distantly related P450 enzymes from Bacillus subtilis or any other organism that are competent cyclopropanation catalysts using similar conditions to wild-type P450 BM3 and highly active P450 BM3 variants. As a non-limiting example, site-directed recombination of three bacterial cytochrome P450s can be performed with sequence crossover sites selected to minimize the number of disrupted contacts within the protein structure. In some embodiments, seven crossover sites can be chosen, resulting in eight sequence blocks. One skilled in the art will understand that the number of crossover sites can be chosen to produce the desired number of sequence blocks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 crossover sites for 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequence blocks, respectively. In other embodiments, the numbering used for the chimeric P450 refers to the identity of the parent sequence at each block. For example, 12312312 refers to a sequence containing block 1 from P450 #1, block 2 from P450 #2, block 3 from P450 #3, block 4 from P450 #1, block 5 from P450 #2, and so on. A chimeric library useful for generating the chimeric heme enzymes of the invention can be constructed as described in, e.g., Otey et al., PLoS Biology, 4(5):e112 (2006), following the SISDC method (see, Hiraga et al., J. Mol. Biol., 330:287-96 (2003)) using the type IIb restriction endonuclease BsaXI, ligating the full-length library into the pCWori vector and transforming into the catalase-deficient E. coli strain SN0037 (see, Nakagawa et al., Biosci. Biotechnol. Biochem., 60:415-420 (1996)); the disclosures of these references are hereby incorporated by reference in their entirety for all purposes.
[0144] As a non-limiting example, chimeric P450 proteins comprising recombined sequences or blocks of amino acids from CYP102A1 (Accession No. J04832), CYP102A2 (Accession No. CAB12544), and CYP102A3 (Accession No. U93874) can be constructed. In certain instances, the CYP102A1 parent sequence is assigned 1, the CYP102A2 parent sequence is assigned 2, and the CYP102A3 is parent sequence assigned 3. In some instances, each parent sequence is divided into eight sequence blocks containing the following amino acids (aa): block 1: aa 1-64; block 2: aa 65-122; block 3: aa 123-166; block 4: aa 167-216; block 5: aa 217-268; block 6: aa 269-328; block 7: aa 329-404; and block 8: aa 405-end. Thus, in this example, there are eight blocks of amino acids and three fragments are possible at each block. For instance, 12312312 refers to a chimeric P450 protein of the invention containing block 1 (aa 1-64) from CYP102A1, block 2 (aa 65-122) from CYP102A2, block 3 (aa 123-166) from CYP102A3, block 4 (aa 167-216) from CYP102A1, block 5 (aa 217-268) from CYP102A2, and so on. See, e.g., Otey et al., PLoS Biology, 4(5):e112 (2006). Non-limiting examples of chimeric P450 proteins include those set forth in Table 5B (C.sub.2G9, X7, X7-12, C2E6, X7-9, C2B12, TSP234). In some embodiments, the chimeric heme enzymes of the invention can comprise at least one or more of the mutations described herein.
[0145] In some embodiments, the present invention provides the incorporation of homologous or analogous mutations to C400X (AxX) and/or T268A in other cytochrome P450 enzymes and heme enzymes in order to impart or enhance cyclopropanation activity.
[0146] As non-limiting examples, the cytochrome P450 can be a variant of CYP101A1 (SEQ ID NO:25) comprising a C357X (e.g., C357S) mutation, a T252A mutation, or a combination of C357X (e.g., C357S) and T252A mutations, wherein X is any amino acid other than Cys, or the cytochrome P450 can be a variant of CYP2B4 (SEQ ID NO:28) comprising a C436X (e.g., C436S) mutation, a T302A mutation, or a combination of C436X (e.g., C436S) and T302A mutations, wherein X is any amino acid other than Cys, or the cytochrome P450 can be a variant of CYP2D7 (SEQ ID NO:26) comprising a C461X (e.g., C461 S) mutation, wherein X is any amino acid other than Cys, or the cytochrome P450 can be a variant of P450C27 (SEQ ID NO:27) comprising a C478X (e.g., C478S) mutation, wherein X is any amino acid other than Cys.
[0147] In other embodiments, the heme protein is a cytochrome c or a variant thereof. In a particular embodiment, the heme protein is a mature cytochrome c protein (residues 29-152 of the unprocessed peptide) B3FQS5_RHOMR (Swiss-Prot: B3FQS5) from Rhodothermus marinus (Rhodothermus obamensis) or a variant thereof (Biochemistry 2008, 47, 11953-11963). In a further embodiment, the B3FQS5_RHOMR protein (Rma cyt c) contains a mutation at the axial heme ligand residue M100 (mature peptide numbering convention) to any other amino acid residue that is among the naturally occurring twenty amino acids. In a further embodiment, the Rma cyt c protein consists of a single mutation of the position V75 to any other amino acid. In a further embodiment, the Rma cyt c protein consists of any combination of mutations residues M100 and V75 to any other amino acid.
[0148] In some embodiments, the heme protein is a cytochrome c protein or a variant thereof. In a particular embodiment, the heme protein is a cytochrome c protein CYC2_RHOGL (Swiss-Prot: P00080) from Rhodopila globiformis (Rhodopsuedomonas globiformis) or a variant thereof (Arch. Biochem. Biophys. 1996, 333, 338-348). In a particular embodiment, the heme protein is a mature cytochrome c protein (residues 19-98 of the unprocessed peptide) CY552_HYDTT (Swiss-Prot: P15452) from Hydrogenobacter thermophilus (strain DSM 6534/IAM 12695/TK-6) or a variant thereof (J. Biol. Chem. 2005, 280, 25729-25734). In a further embodiment, the CY552_HYDTT protein (Hth cyt c) contains a mutation at the axial heme ligand residue M59 (mature peptide numbering convention) to any other amino acid residue that is among the naturally occurring twenty amino acids. In a further embodiment, the Hth cyt c protein consists of a single mutation of the position Q62 to any other amino acid. In a further embodiment, the Hth cyt c protein consists of any combination of mutations residues M59 and Q62 to any other amino acid.
[0149] In certain embodiments, the heme protein is a globin or a variant thereof. In a particular embodiment, the heme protein is a Hell's Gate globin B3DUZ7_METI4 (Swiss-Prot: B3DUZ7) from Methylacidiphilum infernorum (Methylokorus infernorum) or a variant thereof. In a further embodiment, the Hell's Gate globin (HGG) contains a mutation at residue Y29 to any other amino acid residue that is among the naturally occurring twenty amino acids. In a further embodiment, the HGG protein consists of a single mutation of the position Q50 to any other amino acid. In a further embodiment, the HGG protein consists of any combination of mutations residues Y29 and Q50 to any other amino acid.
[0150] In some embodiments, the globin is myoglobin or a variant thereof. In some embodiments, the globin is M. infernorum hemoglobin according to SEQ ID NO:61 or a variant thereof. In some embodiments, the M. infernorum hemoglobin variant comprises one or more mutations of amino acid residues selected from the group consisting of F28, Y29, L32, L54, and V95. In some embodiments, the M. infernorum hemoglobin variant comprises one or more mutations selected from the group consisting of F28S, Y29A, L32A, L32C, L32T, L54S, and V95F. In some such embodiments, the M. infernorum variant comprises a V95F mutation.
[0151] In some embodiments, the globin is B. subtilis truncated hemoglobin according to SEQ ID NO:62 or a variant thereof. In some embodiments, the B. subtilis hemoglobin variant comprises one or more mutations of amino acid residues selected from the group consisting of T45 and Q49. In some embodiments, the B. subtilis hemoglobin variant comprises a T45 mutation and a Q49 mutation. In some embodiments, the B. subtilis hemoglobin variant comprises one or more mutations selected from the group consisting of T45L, T45F, T45A, Q49L, Q49F, and Q49A. In some such embodiments, the B. subtilis hemoglobin variant comprises a first mutation selected from the group consisting of T45L, T45F, and T45A, and a second mutation selected from the group consisting of Q49L, Q49F, and Q49A.
[0152] In some embodiments, the heme protein is a myoglobin or a variant thereof. In a particular embodiment, the heme protein is sperm whale myoglobin or a variant thereof. In a further embodiment, the myoglobin protein (Mb) contains a mutation at residue H64 to any other amino acid residue that is among the naturally occurring twenty amino acids. In a further embodiment, the Mb protein contains a single mutation of the position V68 to any other amino acid. In a further embodiment, the Mb protein contains any combination of mutations of residues M64 and V68 to any other amino acid.
[0153] In some embodiments, the heme protein is a peroxidase or a variant thereof. In some embodiments, the heme protein is a catalase or a variant thereof.
[0154] An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the heme enzymes of the invention range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present invention have higher TTNs compared to the wild-type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro cyclopropanation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell cyclopropanation reactions.
[0155] In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active cyclopropanation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo cyclopropanation reactions of the present invention. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400X mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes.
[0156] When whole cells expressing a heme enzyme are used to carry out a cyclopropanation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo cyclopropanation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol.Math.g.sub.cdw.sup.1, wherein g.sub.cdw is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol.Math.g.sub.cdw.sup.1, or from about 1 to about 10 mmol.Math.g.sub.cdw.sup.1, or from about 5 to about 10 mmol.Math.g.sub.cdw.sup.1, or from about 0.01 to about 1 mmol.Math.g.sub.cdw.sup.1, or from about 0.01 to about 0.1 mmol.Math.g.sub.cdw.sup.1, or from about 0.1 to about 1 mmol.Math.g.sub.cdw.sup.1, or greater than 1 mmol.Math.g.sub.cdw.sup.1. The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol.Math.g.sub.cdw.sup.1.
[0157] When whole cells expressing a heme enzyme are used to carry out a cyclopropanation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g g.sub.cdw.sup.1 h.sup.1). In general, in vivo cyclopropanation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g.Math.g.sub.cdw.sup.1 h.sup.1, wherein g.sub.cdw is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g g.sub.cdw.sup.1 h.sup.1, or from about 0.1 to about 0.5 g g.sub.cdw.sup.1 h.sup.1, or greater than 0.5 g g.sub.cdw.sup.1 h.sup.1. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g g.sub.cdw.sup.1 h.sup.1.
[0158] In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce the heme enzymes (e.g., cytochrome P450 variants) of the present invention. The mutated gene can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Cyclopropanation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.
[0159] The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the invention can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.
[0160] The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.
[0161] It is understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST).
[0162] Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE vectors (Qiagen), pBluescript vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors, pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New England Biolabs), pQE vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.
[0163] The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.
[0164] Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5, DH10, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NSO hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.
[0165] In another aspect, the invention provides methods for preparing cyclopropanation products using M. infernorum hemoglobin or B. subtilis hemoglobin, and variants thereof, as catalysts. In some embodiments, the method includes: (al) providing an olefinic substrate, a diazo reagent, and M. infernorum hemoglobin, or a variant thereof; and (b1) admixing the components of step (al) in a reaction for a time sufficient to produce a cyclopropanation product. In some embodiments, the method includes: (a2) providing an olefinic substrate, a diazo reagent, and B. subtilis hemoglobin, or a variant thereof; and (b2) admixing the components of step (a2) in a reaction for a time sufficient to produce a cyclopropanation product.
[0166] In some embodiments, the cyclopropanation product is a compound according to Formula L:
##STR00023##
wherein: [0167] R.sup.11a is independently selected from the group consisting of H, optionally substituted C.sub.1-18 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR.sup.11b, C(O)N(R.sup.17a).sub.2, C(O)R.sup.18a, C(O)C(O)OR.sup.18a, and Si(R.sup.18a).sub.3; [0168] R.sup.12a is independently selected from the group consisting of H, optionally substituted C.sub.1-18 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted 6- to 10-membered heteroaryl, halo, cyano, C(O)OR.sup.12b, C(O)N(R.sup.17).sub.2, C(O)R.sup.18a, C(O)C(O)OR.sup.18a, and Si(R.sup.18a).sub.3; [0169] wherein: [0170] R.sup.11b and R.sup.12b are independently selected from the group consisting of H, optionally substituted C.sub.1-18 alkyl and -L-R.sup.C, wherein [0171] each L is selected from the group consisting of a bond, C(R.sup.L).sub.2, and NR.sup.LC(R.sup.L).sub.2, [0172] each R.sup.L is independently selected from the group consisting of H, C.sub.1-6 alkyl, halo, CN, and SO.sub.2, and [0173] each R.sup.C is selected from the group consisting of optionally substituted C.sub.6-10 aryl, optionally substituted 6- to 10-membered heteroraryl, and optionally substituted 6- to 10-membered heterocyclyl; and [0174] R.sup.13a, R.sup.14a, R.sup.15a, and R.sup.16a are independently selected from the group consisting of H, C.sub.1-18 alkyl, C.sub.2-18 alkenyl, C.sub.2-18 alkynyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-C.sub.6 alkoxy, halo, hydroxy, cyano, C(O)N(R.sup.17a).sub.2, NR.sup.17aC(O)R.sup.18a, C(O)R.sup.18a, C(O)OR.sup.18a, and N(R.sup.19a).sub.2, [0175] wherein: [0176] each R.sup.17a and R.sup.18a is independently selected from the group consisting of H, optionally substituted C.sub.1-12 alkyl, optionally substituted C.sub.2-12 alkenyl, and optionally substituted C.sub.6-10 aryl; and [0177] each R.sup.19a is independently selected from the group consisting of H, optionally substituted C.sub.6-10 aryl, and optionally substituted 6- to 10-membered heteroaryl, or two R.sup.19a moieties, together with the nitrogen atom to which they are attached, can form 6- to 18-membered heterocyclyl; [0178] or R.sup.13a forms an optionally substituted 3- to 18-membered ring with R.sup.4; [0179] or R.sup.15a forms an optionally substituted 3- to 18-membered ring with R.sup.6; [0180] or R.sup.13a or R.sup.14a forms a double bond with R.sup.15a or R16a; [0181] or R.sup.13a or R.sup.14a forms an optionally substituted 5- to 6-membered ring with R.sup.15a or R.sup.16a.
[0182] M. infernorum hemoglobin and B. subtilis hemoglobin, or variants thereof, can be used for preparing a number of cyclopropanation products including, but not limited to, commodity and fine chemicals, flavors and scents, insecticides, and active ingredients in pharmaceutical compositions. The cyclopropanation products can also serve as starting materials or intermediates for the synthesis of compounds belonging to these and other classes. For example, M. infernorum hemoglobin, B. subtilis hemoglobin, and variants thereof can be used in the methods of the invention for preparation of pyrethroids, milnacipran, bicifidine, cilastain, boceprevir, sitafloxacin, sitafloxacin, anthoplalone, noranthoplone, odanacatib, montekulast, montekulast. These and other cyclopropanation products are described, for example, in U.S. Pat. No. 8,993,262, which is incorporated herein by reference in its entirety.
[0183] B. Cyclopropanation Substrates
[0184] In some embodiments, the invention provides a method for producing a cyclopropanation product of Formula A:
##STR00024##
wherein R.sup.6 is C.sub.1-18 alkoxy. The method includes combining an olefinic substrate, a diazoester carbene precursor, and a heme enzyme under conditions sufficient to form the product of Formula A.
[0185] In some embodiments, the cyclopropanation product is a compound of Formula XVII:
##STR00025## [0186] the olefinic substrate is a compound of Formula V
##STR00026##
and [0187] the carbene precursor is a compound of Formula XVI,
##STR00027## [0188] wherein R.sup.6a is C.sub.1-18 alkyl.
[0189] In some embodiments, the cyclopropanation product is a compound according to Formula XVIIa:
##STR00028##
[0190] In some embodiments, R.sup.6a is selected from the group consisting of C.sub.1-8 alkyl, C.sub.1-12 alkyl, C.sub.1-6 alkyl, and C.sub.1-4 alkyl. In some embodiments, R.sup.6a is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. In some embodiments, R.sup.6a is ethyl. In some embodiments, the cyclopropanation product is a compound according to Formula VIIa:
##STR00029##
[0191] In particular embodiments, the present invention provides a method for the synthesis of trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine catalyzed by a heme enzyme such as a cytochrome P450 enzyme (e.g., P450 BM3 enzyme) using a diazoketone as the carbene precursor and the Beckmann rearrangement as shown in Scheme 5 above, wherein R is an optionally substituted C.sub.1-18 alkyl, alkenyl, or alkynyl, or an optionally substituted C.sub.6-10 aryl or heteroaryl.
[0192] In certain aspects, the methods of the present invention for the enzymatic synthesis of trans-(1R,2S)-2-(3,4-difluorophenyl)-cyclopropylamine comprises incubating an olefinic substrate such as a styrene and a carbene precursor such as a diazoketone reagent with a cyclopropanation catalyst such as a heme enzyme to form a cyclopropane product.
[0193] In some embodiments, the styrene has a structure according to Formula XXX:
##STR00030##
wherein R.sup.21 is selected from H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, C(O)N(R.sup.27).sub.2, C(O)OR.sup.28, N(R.sup.29).sub.2, halo, hydroxy, and cyano. R.sup.22 and R.sup.23 are independently selected from H, optionally substituted C.sub.1-6 alkyl, and halo. R.sup.24 is selected from optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, halo, and haloalkyl, and the subscript r is an integer from 0 to 2.
[0194] In particular embodiments, R.sup.21, R.sup.22 and R.sup.23 are all H, R.sup.24 is a halogen such as fluorine, and r is 2. In preferred embodiments, the styrene is 1,2-difluoro-4-vinylbenzene (i.e., 3,4-difluorostyrene).
[0195] In some embodiments, the diazoketone a structure according to Formula XXXI:
##STR00031##
wherein R.sup.25 is selected from an optionally substituted C.sub.1-18 alkyl, alkenyl, or alkynyl, or an optionally substituted C.sub.6-10 aryl or heteroaryl.
[0196] Accordingly, some embodiments of the invention provide a method for producing a cyclopropanation product of Formula A:
##STR00032##
wherein R.sup.6 is selected from the group consisting of C.sub.1-18 alkyl, C.sub.1-18 alkenyl, and C.sub.1-18 alkynyl. The method includes combining an olefinic substrate, a diazoketone carbene precursor, and a heme enzyme under conditions sufficient to form the product of Formula A.
[0197] In some embodiments, the cyclopropanation product is a compound of Formula XXVII:
##STR00033## [0198] the olefinic substrate is a compound of Formula V:
##STR00034##
and [0199] the carbene precursor is a compound of Formula XXVI:
##STR00035## [0200] wherein R.sup.6b is C.sub.1-18 alkyl.
[0201] In some embodiments, the cyclopropanation product is a compound according to Formula XXVIIa:
##STR00036##
[0202] In some embodiments, R.sup.6b is selected from the group consisting of C.sub.1-8 alkyl, C.sub.1-12 alkyl, C.sub.1-6 alkyl, and C.sub.1-4 alkyl. In some embodiments, R.sup.6b is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. In some embodiments, R.sup.6b is methyl.
[0203] One of skill in the art will appreciate that the stereochemical configuration of the cyclopropanation product will be determined in part by the orientation of the carbene precursor reagent (i.e., the diazoester or the diazoketone) with respect to the position of an olefinic substrate such as styrene during the cyclopropanation step. For example, any substituent originating from the olefinic substrate can be positioned on the same side of the cyclopropyl ring as a substituent originating from the carbene precursor reagent. Cyclopropanation products having this arrangement are called cis compounds or Z compounds. Any substituent originating from the olefinic substrate and any substituent originating from the carbene precursor reagent can also be on opposite sides of the cyclopropyl ring. Cyclopropanation products having this arrangement are called trans compounds or E compounds.
[0204] Two cis isomers and two trans isomers can arise from the reaction of an olefinic substrate with a carbene precursor reagent. The two cis isomers are enantiomers with respect to one another, in that the structures are non-superimposable mirror images of each other. Similarly, the two trans isomers are enantiomers. One of skill in the art will appreciate that the absolute stereochemistry of a cyclopropanation productthat is, whether a given chiral center exhibits the right-handed R configuration or the left-handed S configurationwill depend on factors including the structures of the particular olefinic substrate and carbene precursor reagent used in the reaction, as well as the identity of the enzyme. This is also true for the relative stereochemistrythat is, whether a cyclopropanation product exhibits a cis or trans configurationas well as for the distribution of cyclopropanation product mixtures will also depend on such factors.
[0205] In general, cyclopropanation product mixtures have cis:trans ratios ranging from about 1:99 to about 99:1. The cis:trans ratio can be, for example, from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or from about 1:50 to about 1:25, or from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The cis:trans ratio can be from about 1:80 to about 1:20, or from about 1:60 to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The cis:trans ratio can be about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or about 1:95. The cis:trans ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.
[0206] The distribution of a cyclopropanation product mixture can be assessed in terms of the enantiomeric excess, or % ee, of the mixture. The enantiomeric excess refers to the difference in the mole fractions of two enantiomers in a mixture. The enantiomeric excess of the E or trans (R,R) and (S,S) enantiomers, for example, can be calculated using the formula: % ee.sub.E=[(.sub.R,R.sub.S,S)/(.sub.R,R+.sub.S,S)]100%, wherein is the mole fraction for a given enantiomer. The enantiomeric excess of the Z or cis enantiomers (% ee.sub.Z) can be calculated in the same manner.
[0207] In general, cyclopropanation product mixtures exhibit % ee values ranging from about 1% to about 99%, or from about 1% to about 99%. The closer a given % ee value is to 99% (or 99%), the purer the reaction mixture is. The % ee can be, for example, from about 90% to about 90%, or from about 80% to about 80%, or from about 70% to about 70%, or from about 60% to about 60%, or from about 40% to about 40%, or from about 20% to about 20%. The % ee can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The % ee can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The % ee can be about 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95%. Any of these values can be % ee.sub.E values or % ee.sub.Z values.
[0208] Accordingly, some embodiments of the invention provide methods for producing a plurality of cyclopropanation products having a % ee.sub.Z of from about 90% to about 90%. In some embodiments, the % ee.sub.Z is at least 90%. In some embodiments, the % ee.sub.Z is at least 99%. In some embodiments, the % ee.sub.E is from about 90% to about 90%. In some embodiments, the % ee.sub.E is at least 90%. In some embodiments, the % ee.sub.E is at least 99%.
[0209] C. Reaction Conditions
[0210] The methods of the invention include forming reaction mixtures that contain the heme enzymes described herein. The heme enzymes can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a heme enzyme can catalyze the reaction within a cell expressing the heme enzyme. Any suitable amount of heme enzyme can be used in the methods of the invention. In general, cyclopropanation reaction mixtures contain from about 0.01 mol % to about 10 mol % heme enzyme with respect to the diazo reagent (e.g., diazoketone) and/or olefinic substrate. The reaction mixtures can contain, for example, from about 0.01 mol % to about 0.1 mol % heme enzyme, or from about 0.1 mol % to about 1 mol % heme enzyme, or from about 1 mol % to about 10 mol % heme enzyme. The reaction mixtures can contain from about 0.05 mol % to about 5 mol % heme enzyme, or from about 0.05 mol % to about 0.5 mol % heme enzyme. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1 mol % heme enzyme.
[0211] The concentration of olefinic substrate and carbene precursor reagent are typically in the range of from about 100 M to about 1 M. The concentration can be, for example, from about 100 M to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 M to about 500 mM, 500 M to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of olefinic substrate or carbene precursor reagent can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 M. The concentration of olefinic substrate or carbene precursor reagent can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.
[0212] Cyclopropanation reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KCl, CaCl.sub.2, and salts of Mn.sup.2+ and Mg.sup.2+), denaturants (e.g., urea and guanidinium hydrochloride), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), -mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 M to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 M, or about 10 M, or about 100 M, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. In some embodiments, a reducing agent is used in a sub-stoichiometric amount with respect to the olefin substrate and the carbene precursor reagent. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A cosolvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v).
[0213] Reactions are conducted under conditions sufficient to catalyze the formation of a cyclopropanation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4 C. to about 40 C. The reactions can be conducted, for example, at about 25 C. or about 37 C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 6 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours. Reactions can be conducted under aerobic conditions or anaerobic conditions. Reactions can be conducted under an inert atmosphere, such as a nitrogen atmosphere or argon atmosphere. In some embodiments, a solvent is added to the reaction mixture. In some embodiments, the solvent forms a second phase, and the cyclopropanation occurs in the aqueous phase. In some embodiments, the heme enzyme is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the invention, depending on the identity of a particular heme enzyme, olefinic substrate, or carbene precursor reagent.
[0214] Reactions can be conducted in vivo with intact cells expressing a heme enzyme of the invention. The in vivo reactions can be conducted with any of the host cells used for expression of the heme enzymes, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Cyclopropanation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for cyclopropanation reactions. Other densities can be useful, depending on the cell type, specific heme enzymes, or other factors.
[0215] The methods of the invention can be assessed in terms of the diastereoselectivity and/or enantioselectivity of the cyclopropanation reactionthat is, the extent to which the reaction produces a particular isomer, whether a diastereomer or enantiomer. A perfectly selective reaction produces a single isomer, such that the isomer constitutes 100% of the product. As another non-limiting example, a reaction producing a particular enantiomer constituting 90% of the total product can be said to be 90% enantioselective. A reaction producing a particular diastereomer constituting 30% of the total product, meanwhile, can be said to be 30% diastereoselective.
[0216] In general, the methods of the invention include reactions that are from about 1% to about 99% diastereoselective. The reactions are from about 1% to about 99% enantioselective. The reaction can be, for example, from about 10% to about 90% diastereoselective, or from about 20% to about 80% diastereoselective, or from about 40% to about 60% diastereoselective, or from about 1% to about 25% diastereoselective, or from about 25% to about 50% diastereoselective, or from about 50% to about 75% diastereoselective. The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% diastereoselective. The reaction can be from about 10% to about 90% enantioselective, from about 20% to about 80% enantioselective, or from about 40% to about 60% enantioselective, or from about 1% to about 25% enantioselective, or from about 25% to about 50% enantioselective, or from about 50% to about 75% enantioselective. The reaction can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or about 95% enantioselective. Accordingly some embodiments of the invention provide methods wherein the reaction is at least 30% to at least 90% diastereoselective. In some embodiments, the reaction is at least 30% to at least 90% enantioselective.
[0217] As described above, some embodiments of the invention provide methods that include: a) reacting a benzaldehyde of Formula XLIII with a Wittig reagent of Formula XLIV in the presence of a first base in a first solvent to produce a substituted styrene of Formula XLV; b) reacting the styrene of Formula XLV with a diazoester compound of Formula XLVI in the presence of a heme protein catalyst to produce a cyclopropanecarboxylate of Formula XLVIIa; c) hydrolyzing the cyclopropanecarboxylate of Formula XLVIIa with an acid or a second base in a third solvent to produce a cyclopropanecarboxylic acid of Formula XLVIIIa; and f) reacting the cyclopropanecarboxylic acid compound of Formula XLVIIIa with an azide compound in the presence a third base in a fifth solvent to produce an isocyanate intermediate.
[0218] Exemplary first solvents used in step-(a) include, but are not limited to, an ester, a nitrile, a hydrocarbon, a cyclic ether, an aliphatic ether, a polar aprotic solvent, and mixtures thereof. The term solvent also includes mixtures of solvents.
[0219] Specifically, the first solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, isobutyl acetate, tert-butyl acetate, acetonitrile, propionitrile, tetrahydrofuran, 2-methyl-tetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, diethyl ether, diisopropyl ether, monoglyme, diglyme, n-hexane, n-heptane, cyclohexane, toluene, xylene, N,N-dimethylformamide, N,N-dimethylacetamide, dimethylsulfoxide, N-methylpyrrolidone, and mixtures thereof; and a most specific solvent is toluene.
[0220] In one embodiment, the first base used in step-(a) is an organic or inorganic base. Exemplary organic bases include, but are not limited to, alkyl metals such as methyl lithium, butyl lithium, hexyllithium; alkali metal complexes with amines such as lithium diisopropyl amide; and organic amine bases of formula NR.sup.101R.sup.102R.sup.103, wherein R.sup.101, R.sup.102, and R.sup.103 are independently hydrogen, C.sub.1-6 straight or branched chain alkyl, aryl alkyl, or C.sub.3-10 single or fused ring optionally substituted, alkylcycloalkyl; or independently R.sup.101, R.sup.102, and R.sup.103 combine with each other to form a C.sub.3-7 membered cycloalkyl ring or heterocyclic system containing one or more hetero atoms. Specific organic bases are trimethylamine, dimethyl amine, diethylamine, tert-butyl amine, tributylamine, triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, 4-(N,N-dimethylamino)pyridine, methyl lithium, butyl lithium, hexyllithium, lithium diisopropyl amide, 1,8-diazabicyclo[5.4.0]undec-7-ene; and most specifically butyl lithium and 1,8-diazabicyclo[5.4.0]undec-7-ene.
[0221] Exemplary inorganic bases include, but are not limited to, hydroxides, alkoxides, bicarbonates and carbonates of alkali or alkaline earth metals, and ammonia. Specific inorganic bases are aqueous ammonia, sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, lithium carbonate, sodium tert-butoxide, sodium isopropoxide and potassium tert-butoxide, and more specifically sodium tert-butoxide, sodium isopropoxide and potassium tert-butoxide.
[0222] Specific Wittig reagents used in step-(a) are methyl triphenylphosphonium chloride, methyl triphenylphosphonium bromide, methyl triphenylphosphonium iodide, and more specifically methyl triphenylphosphonium bromide.
[0223] In one embodiment, the reaction in step-(a) is carried out at a temperature of about 50 C. to about 150 C. for at least 30 minutes, specifically at a temperature of 0 C. to about 100 C. for about 2 hours to about 10 hours, and more specifically at about 35 C. to about 80 C. for about 3 hours to about 6 hours.
[0224] The reaction mass containing the substituted styrene compound of Formula XLV obtained in step-(a) may be subjected to usual work up such as a washing, an extraction, a pH adjustment, an evaporation or a combination thereof. The reaction mass may be used directly in the next step or the styrene compound of Formula XLV may be isolated and then used in the next step.
[0225] In one embodiment, the styrene compound of Formula XLV is isolated from a suitable solvent by conventional methods such as cooling, seeding, partial removal of the solvent from the solution, by adding an anti-solvent to the solution, evaporation, vacuum distillation, or a combination thereof.
[0226] The reaction mass containing the substituted cyclopropanecarboxylate compound of Formula XLVII obtained in step-(b) may be subjected to usual work up such as a washing, an extraction, a pH adjustment, an evaporation or a combination thereof. The reaction mass may be used directly in the next step to produce the cyclopropanecarboxylic acid compound of Formula XLVIII, or the cyclopropanecarboxylate compound of Formula XLVII may be isolated and then used in the next step.
[0227] In one embodiment, the cyclopropanecarboxylate compound of Formula XLVII is isolated from a suitable solvent by the methods as described above.
[0228] In another embodiment, the solvent used to isolate the cyclopropanecarboxylate compound of Formula XLVII is selected from the group consisting of water, an aliphatic ether, a hydrocarbon solvent, a chlorinated hydrocarbon, and mixtures thereof. Specifically, the solvent is selected from the group consisting of water, toluene, xylene, dichloromethane, diethyl ether, diisopropyl ether, n-heptane, n-pentane, n-hexane, cyclohexane, and mixtures thereof.
EXAMPLES
[0229] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
Example 1. In Vitro Enzymatic Synthesis of Cyclopropanes Using CYP102A1 Variants
[0230] This example illustrates the purification of CYP102A1 (BM3) enzymes and their use in the production of the compounds of Formulae VII or VIIa.
[0231] P450 expression and purification. For the enzymatic transformations, P450 variants were used in purified form. One liter Hyperbroth.sub.amp was inoculated with an overnight culture (25 mL, TB.sub.amp) of recombinant E. coli BL21 cells harboring a pCWori or pET22 plasmid encoding the P450 variant under the control of the tac promoter. The cultures were shaken at 200 rpm at 37 C. for roughly 3.5 h or until an optical of density of 1.2-1.8 was reached. The temperature was reduced to 22 C. and the shake rate was reduced to 130-150 rpm for 20 min, then the cultures were induced by adding IPTG and aminolevulinic acid to a final concentration of 0.25 mM and 0.5 mM respectively. The cultures were allowed to continue for another 20 hours at this temperature and shake rate. Cell were harvested by centrifugation (4 C., 15 min, 3,000g), and the cell pellet was stored at 20 C. or below for at least 2 h. For the purification of 6His tagged P450s, the thawed cell pellet was resuspended in Ni-NTA buffer A (25 mM Tris.HCl, 200 mM NaCl, 25 mM imidazole, pH 8.0, 4 mL/gcw) and lysed by sonication (21 min, output control 5, 50% duty cycle). The lysate was centrifuged at 27,000g for 20 min at 4 C. to remove cell debris. The collected supernatant was first subjected to a Ni-NTA chromatography step using a Ni Sepharose column (HisTrap-HP, GE healthcare, Piscataway, N.J.). The P450 was eluted from the Ni Sepharose column using 25 mM Tris.HCl, 200 mM NaCl, 300 mM imidazole, pH 8.0. Ni-purified protein was buffer exchanged into 0.1 M phosphate buffer (pH=8.0) using a 30 kDa molecular weight cut-off centrifugal filter. Protein concentrations were determined by CO-assay. For storage, proteins were portioned into 300 L aliquots and stored at 80 C.
[0232] Small-Scale In Vitro Protein Reactions (Anaerobic).
[0233] Small-scale (400 L) reactions were carried out in 2 mL glass crimp vials (Agilent Technologies, San Diego, Calif.). P450 solution (60 L, 67 M) was added to an unsealed crimp vial before crimp sealing with a silicone septum. A 12.5 mM solution of sodium dithionite in phosphate buffer (0.1 M, pH=8.0) was degassed by bubbling with argon in a 6 mL crimp-sealed vial. The headspace of the 2 mL vials containing P450 solution were flushed with argon (no bubbling). If multiple reactions were being carried out in parallel, a maximum of 8 vials were connected via cannulae and degassed in series. The buffer/dithionite solution (320 L) was then added to each reaction vial via syringe, and the gas lines were disconnected from the vials. 10 L of a stock solution of olefin (400 mM for styrene of Formula V) was added via a glass syringe, followed by 10 L of a 400 mM stock of ethyldiazoacetate (EDA, example compound of Formula VI) (both stocks in EtOH). The reaction vials were then placed in a tray on a plate shaker and left to shake at 350 rpm for 12 h at room temperature. The final concentrations of the reagents were typically: 10 olefin, 8.7 mM EDA, 10 mM Na.sub.2S.sub.2O.sub.4, and 10 M P450. The reaction was quenched by the addition of 3 M HCl (25 L). The vials were uncapped and 1 mL of cyclohexane was added, followed by 20 L of a 20 mM solution of 2-phenylethanol solution in cyclohexane (internal standard). The mixture was transferred to a 1.5 mL Eppendorf tube and vortexed and centrifuged (10,000rcf, 30 s). The organic layer was then analyzed by supercritical fluid chromatography (SFC).
[0234] The results of the small scale reactions are presented below and demonstrate that a number of CYP102A1 variants are capable of catalyzing formation of the desired cyclopropane carboxylate ethyl ester of Formula VIIa. Specifically, the best variant found in this initial screen of CYP102A1 variants encoded mutations T268A and C400H and gave a modest level of asymmetric induction (18% ee). This can be improved by further engineering, if desired.
TABLE-US-00007 TABLE 6
Example 2. In Vitro Enzymatic Synthesis of Cyclopropanes Using CYP119 Variants
[0235] CYP119 variants was expressed in BL21(DE3). Seed cultures of 2XYT-amp (50 mL, 100 g/mL ampicillin) were inoculated from glycerol stocks and grown overnight (200 RPM, 30 C.) in Erlenmeyer flasks (125 mL capacity). The resulting cultures were used to inoculate 1 L of Hyper Broth supplemented with ampicillin (100 g/mL) in Fembach flasks (2.8 L capacity). After inoculation, cultures were grown at 37 C. and 180 RPM for 3.5 hours, then cooled on ice for 10-15 minutes and then induced via addition of IPTG (0.25 mM final concentration) and aminolevulinic acid (0.5 mM final concentration). Induced cultures were then grown overnight at reduced temperature and agitation rate (140 RPM, 25 C.). Following expression, cells were pelleted and frozen at 20 C. until purification.
[0236] For purification, frozen cell pellets were resuspended (4 mL/g wet cell weight) in lysis buffer (25 mM Tris, 100 mM NaCl, 30 mM imidazole, lysozyme (0.5 mg/mL), DNaseI (0.02 mg/mL), hemin (1 mg/g wet cell weight), pH 7.5). Cells were disrupted by sonication (3 min, output control 1.5, duty cycle: 5 sec on/10 sec off, Sonicator 3000, Misonix, Inc.). The cell suspension was subsequently incubated for 30 min at 65 C. to precipitate E. coli proteins. To pellet insoluble cell debris, lysates were centrifuged (20,000g for 30 min at 4 C.). Cleared lysates were then purified loaded onto Ni-NTA columns (5 mL size, HP resin, GE Healthcare) using an AKTAxpress purifier FPLC system (GE healthcare). Target proteins were eluted using a linear gradient from 100% buffer A (25 mM TRIS-HCL, 100 mM NaCl, 10 mM, pH 7.5), 0% buffer B (25 mM Tris, 100 mM NaCl, 300 mM imidazole pH 7.5) to 100% buffer B over 10 column volumes.
[0237] Proteins were then pooled, concentrated to 1 mL, and subjected to three 10-fold dilution and concentration steps using centrifugal spin filters (Vivaspin 20, 30 kDA molecular weight cut-off, GE healthcare), each time diluting into fresh buffer (0.1 M KPi pH 8.0).
[0238] Small-scale (200 L) reactions were carried out in 1.7 mL Eppendorf tubes (Agilent Technologies, San Diego, Calif.). P450 solution (5 L) was added to an unsealed tube before placing in an anaerobic chamber after several cycles of evacuation and refilling with nitrogen gas. A 10.4 mM solution of sodium dithionite in phosphate buffer (0.1 M, pH=8.0) was made by dissolving solid sodium dithionite that had been previously placed in the anaerobic chamber with degassed 0.1 M KPi buffer. The buffer/dithionite solution (320 L) was then added to each reaction vial in the anaerobic chamber by pipetting. 5 L of a stock solution of olefin (400 mM for styrene of Formula V) was added via a glass syringe, followed by 5 L of a 400 mM stock of ethyldiazoacetate (EDA, example compound of Formula VI) (both stocks in methanol). The reaction tubes were then mixed, and allowed to incubate at room temperature in the anaerobic chamber for 12 h at room temperature. The reaction was quenched by removal from the anaerobic chamber, and immediate addition of 3 M HCl (12.5 L). The vials were uncapped and 0.5 mL of cyclohexane was added, followed by 10 L of a 20 mM solution of 2-phenylethanol solution in cyclohexane (internal standard). The mixture was vortexed and centrifuged (10,000rcf, 30 s). The organic layer was then analyzed by supercritical fluid chromatography (SFC).
[0239] Results of small-scale reactions are presented below and demonstrate that a number of CYP119 variants are capable of catalyzing formation of the desired cyclopropane carboxylate ethyl ester of Formula VIIa. Specifically, the best variant found in this initial screen encoded a single mutation H315S and gave a modest level of asymmetric induction (31% ee). The opposite enantiomer was obtained for cyclopropanation of the styrene starting material with WT-T268A; WT-T268A gave 40% for the undesired enantiomer.
TABLE-US-00008 TABLE 7
Example 3. In Vivo Enzymatic Synthesis of Cyclopropanes Using Cytochrome c, Myoglobin, Globin and P450 Hstar Variants
[0240] Expression of Cytochrome c and Variants Thereof.
[0241] One liter Hyperbroth (100 g/mL ampicillin, 20 g/mL chloramphenicol) was inoculated with an overnight culture of 20 mL LB (100 g/mL ampicillin, 20 g/mL chloramphenicol). The overnight culture contained recombinant E. coli BL21-DE3 cells harboring a pET22 plasmid and pEC86 plasmid, encoding the cytochrome c variant under the control of the T7 promoter, and the cytochrome c maturation (ccm) operon under the control of a tet promoter, respectively. The cultures were shaken at 200 rpm at 37 C. for approximately 2 h or until an optical of density of 0.60.9 was reached. The flask containing the cells was placed on ice for 30 min. The incubator temperature was reduced to 20 C., maintaining the 200 rpm shake rate. Cultures were induced by adding IPTG and aminolevulinic acid to a final concentration of 20 M and 200 M respectively. The cultures were allowed to continue for another 20-24 hours at this temperature and shake rate. Cells were harvested by centrifugation (4 C., 15 min, 3,000g) to produce a cell pellet.
[0242] The procedure for expression of myoglobin (Mb), globin (HGG), BM3 Hstar, and variants thereof was similar to that described for expression of CYP102A1 as described above.
[0243] Preparation of Whole Cell Catalysts.
[0244] To prepare whole cells for catalysis, the cell pellet prepared in the previous paragraphs was resuspended in M9-N minimal media (M9 media without ammonium chloride) to an optical density (OD.sub.600) of 60.
[0245] Small-Scale In Vivo Reactions (Anaerobic).
[0246] Small-scale (400 L) reactions were carried out in 2 mL glass crimp vials (Agilent Technologies, San Diego, Calif.). Whole cell catalysts (340 L, OD.sub.600=60 in M9-N minimal media) were added to an unsealed crimp vial before crimp sealing with a silicone septum. The headspace of the vial was flushed with argon for 10 min (no bubbling). A solution of glucose (40 L, 250 mM) was added, followed by a solution of olefin of Formula V (10 L, 800 mM in EtOH; for example, 3,4-difluorostyrene) and a solution of diazo reagent of Formula VI (10 L, 400 mM in EtOH; for example ethyldiazoacetate, EDA). The reaction vial was left to shake on a plate shaker at 400 rpm for 1 h at room temperature. To quench the reaction, the vial was uncapped and cyclohexane (1 mL) was added, followed by 2-phenylethanol (20 L, 20 mM in cyclohexane) as an internal standard. The mixture was transferred to a 1.5 mL Eppendorf tube and vortexed and centrifuged (14000rcf, 5 min). The organic layer was analyzed by gas chromatography (GC) and supercritical fluid chromatography (SFC).
[0247] Results of small-scale reactions are presented below and demonstrate that a number of hemoproteins are capable of catalyzing formation of the desired cyclopropane carboxylate ethyl ester of Formula VIIa (the numbers in parentheses correspond to reactions carried out at 4 C. overnight instead of room temperature; the reaction catalyzed by HGG Y29V V68A was carried out using whole cell catalyst with an optical density of 30). Specifically, the best variants found are Hth cyt c encoding the mutations M59A and Q62A, and BM3 Hstar heme domain encoding the mutations H92N and H100N.
TABLE-US-00009 TABLE 8
Example 4. Lipase-Catalyzed Resolution of Cyclopropanes Compounds
[0248] Lipase enzymes purchased from commercial suppliers or expressed by suitable microbial hosts from suitable plasmids described herein are resuspended, dissolved, or otherwise added to reaction mixtures containing cyclopropane carboxylate compounds of Formulae VII or VIIa in a buffer or solvent mixture similar or identical to those described herein for the purposes of carrying out hemoprotein reactions.
[0249] A lipase isolated from Thermomyces lanuginosus (ALMAC lipase kit; AH-45) is added (5% w/v) to a buffered reaction mixture (0.1 M KPi pH 8.0) containing cyclopropane carboxylate esters of Formulae VII or VIIa.
[0250] The resulting suspension/solution is then agitated via magnetic stirring at 500 RPM for 12-16 hours at room temperature.
[0251] After 12-16 hours, a TLC or HPLC sample is taken to verify that the reaction has produced the desired outcome, namely the hydrolysis of the undesired (S, S) esters of formulae VIIb-d, leading to cyclopropane carboxylic acids VIIIb-d (or salts therefrom).
##STR00044##
Example 5. Improvement of CYP102A1 Variant-Catalyzed Synthesis of Cyclopropane Compounds
[0252] Initial experiments focused on variants of P450-BM3 bearing His, Met, Tyr, or Ala at the proximal position. This set represents a range of possible coordinating heteroatom ligands, and His, Met, and Tyr are found at the axial position of naturally occurring heme proteins such as horseradish peroxidase (HRP), cytochrome c, and catalase. Ala was chosen as well because it is an archetypal small amino acid and may allow a water molecule or hydroxide ion to coordinate to the Fe center. To examine how the different axial ligands affect cyclopropanation activity, each of the four axial mutations were introduced into a P450-BM3 holoenzyme containing the additional mutation T268A. This mutation was previously found to be highly beneficial for cyclopropanation. Four variants, T268A-axX (where X denotes the single-letter amino acid code of each axial variant), were expressed as the His-tagged heme domains and purified.
[0253] When the reactions of whole E. coli cells expressing the four P450-BM3 variants with olefin of Formula V and ethyl diazoacetate of Formula VI were monitored, it was determined that a CYP102A1 (BM3) variant encoding mutations T268A and C400H (variant T268A-AxH) gave the most of the desired cyclopropane of Formula VIIa.
[0254] To create a catalyst more enantioselective than T268A-axH, site-saturation mutagenesis is performed at four active-site positions that had been shown previously to affect selectivity in cyclopropanation or monooxygenation; F87, I263, L437 and T438. The libraries are screened in 96-well plates with whole cells and an oxygen quenching system containing glucose oxidase and catalase in sealed plates. The enantioselectivity of each reaction is determined by chiral supercritical fluid chromatography.
[0255] After isolating the most active and enantioselective catalysts from this first round of screening, the catalysts are then subjected to a second round of site-saturation mutagenesis at the positions V78 and L181. The libraries are screened in 96-well plates with whole cells and an oxygen quenching system containing glucose oxidase and catalase in sealed plates. The enantioselectivity of each reaction is determined by chiral supercritical fluid chromatography. The catalysts with the highest activity and enantioselectivity are then chosen for production of the cyclopropane carboxylate ethyl ester of Formula VIIa.
Example 6. Synthesis of Cyclopropane Compounds Using Myoglobin Variants
[0256] Small-scale (400 L) reactions were carried out in 2 mL glass crimp vials (Agilent Technologies, San Diego, Calif.). Myoglobin or hemin was added to an unsealed crimp vial before crimp sealing with a silicone septum. A 12.5 mM solution of sodium dithionite in phosphate buffer (0.1 M, pH=8.0) was degassed by bubbling with argon in a 6 mL crimp-sealed vial. The headspace of the 2 mL vials containing myoglobin or hemin solution were flushed with argon (no bubbling). The buffer/dithionite solution (300 L) was then added to each reaction vial via syringe, and the gas lines were disconnected from the vials. 10 L of a stock solution of olefin (400 mM of 3,4,-difluorostyrene) was added via a glass syringe, followed by 10 L of a 400 mM stock of ethyldiazoacetate (EDA) (Both stocks in EtOH). The reaction vials were then placed in a tray on a plate shaker and left to shake at 350 rpm for 12 h at room temperature. The final concentrations of the reagents were typically: 10 mM olefin, 8.7 mM EDA, 10 mM Na.sub.2SO.sub.4, and 10 M myoglobin or 100 M hemin. The reaction was quenched by the addition of 3 M HCl (25 L). The vials were uncapped and 1 mL of cyclohexane was added, followed by 20 L of a 20 mM solution of 2-phenylethanol solution in cyclohexane (internal standard). The mixture was transferred to a 1.5 mL Eppendorf tube and vortexed and centrifuged (10,000g, 30 s). the organic layer was then analyzed by supercritical fluid chromatography (SFC).
[0257] The results of small-scale reactions containing myoglobin or hemin are presented in Table 9. The results demonstrate that either of these is an efficient catalysts of compounds of Formulae VIIa-d. The opposite enantiomer resulting from styrene cyclopropanation was observed when WT-T-268A was used; WT-T268A gave 40% ee for the undesired enantiomer. These results demonstrate that myoglobin and variants thereof are effective asymmetric cyclopropanation catalysts for compounds of Formula VII, though in this specific examples myoglobin preferentially synthesized enantiomer VIIb rather the preferred enantiomer VIIa. Nevertheless it is clearly shown that what is necessary for an enantioselective cyclopropanation catalyst is a chiral protein scaffold and a heme cofactor.
TABLE-US-00010 TABLE 9
Example 7. Creation of Variant Library for the Hemoglobin I from Methylacidophilum infernorum
[0258] Based on analysis of the crystal structure of the hemoglobin from Methylacidophilum infernorum (GenBank Accession No. ACD83144), 10 amino acid residues were identified in the distal binding pocket for site-saturation mutagenesis. The 10 amino acids were F28, Y29, L32, F43, Q44, N45, Q50, K53, L54 and V95. Site-saturation mutagenesis was carried out on each of these 10 sites according to the following procedure.
[0259] Forward and reverse mutagenic primer pairs were designed to generate 20 different amino acids for each of the 10 amino acid positions. The primers were synthesized and normalized as 5 nmoles by Integrated DNA Technologies, Inc. The primers were diluted with deionized sterile H.sub.2O to approximately 7 M concentration. A PCR reaction was set up in a total volume of 20 l, with the final concentrations as follows: 1 U of Pfu turbo DNA polymerase, 1 Pfu turbo buffer, 0.1 mM of dNTP, 20 ng of template DNA and 0.35 M of forward and reverse primers. The PCR mixture was heated at 95 C. for 5 minutes, then run on 18 cycles of three steps; i) 3 minutes at 95 C., ii) 1 minute at 65 C. and iii) 15 minutes at 68 C., followed by 10 minutes incubation at 72 C. As a template DNA, pET22b vector containing HGbI gene was used. After the PCR reaction was completed, 1 l of FastDigest DpnI from ThermoFisher Scientific was added to digest the template DNA; incubation was for 3 hours at 37 C. Transformation of the variant DNA library into E. coli was accomplished using NEB 5-alpha (E coli DH5Alpha) chemically competent cells. For each transformation 3 l of DNA mixture were used. The three random colonies were picked from each transformation plate and inoculated and grown overnight for submission for Rolling Circle Amplification (RCA) sequencing by Laragen, Inc. to confirm DNA sequences with target mutation(s). Plasmids with the sequences of interest were prepared and used for transformation of E. cloni EXPRESS BL21(DE3) competent cells purchased from Lucigen Corp. to generate bacterial colonies expressing the HGbI variants.
Example 8. Preparation of Biocatalysts for Screening of Site-Saturation Variants of Methylacidophilum infernorum
[0260] A single colony expressing a HGbI variant was inoculated in 1.5 ml of AthenaES hyper broth media containing carbenicillin (100 g/ml) per well of Axygen Scientific 96-well Deep Well plate. The cells were grown in an INFORS HT plate shaker at 37 C. with 1,000 rpm until optical density (OD.sub.600) of 1.0 was reached. The expression of the HGbI variants was induced by adding 3 mM of aminolevulinic acid (ALA) and 3 mM Isopropyl -D-1-thiogalactopyranoside (IPTG), followed by incubation in the INFORS HT plate shaker at 37 C., shaking at 1,000 rpm for 22 hours. To measure the amount of in vivo protein expression, a CO-binding assay was carried out on whole cells. First, microtiter plates containing 200 l of induced cells per well were centrifuged at 3,000 rpm for 15 minutes, and the cell pellet was resuspended in 200 l of buffer (100 mM Kpi buffer, pH 7). Subsequently, each sample was transferred to microtiter plate and absorbance spectra were measured at a wavelength between 400 nm and 500 nm using a Tecan Infinite M200Pro reader. Then the microtiter plate was incubated in a CO-chamber at 2 PSI for 1 hour in a fume hood. The CO-chamber was placed under vacuum and flushed with CO gas twice before incubating. The absorbance spectra were re-measured at a wavelength between 400 nm and 500 nm and recorded again. The remaining cells were centrifuged to form a pellet using a Beckman Coulter AVANTI JXN-26 centrifuge at 5,000 rpm for 15 minutes. After discarding the supernatant, the 96-well plate was transferred to an anaerobic chamber from Coy Laboratory Products Inc. Then 190 l of M9 media that had been pre-purged with nitrogen was added to each well and the cell pellets were resuspended by mixing using an Eppendorf MixMate Vortex Mixer at 1,500 rpm for 3 minutes. The cell suspensions were screened at this stage for cyclopropanation activity.
Example 9. Screening of Variants of the Hemoglobin I from Methylacidophilum infernorum for Cyclopropanation Activity for the Reaction of 3,4-Difluorostyrene and Ethyl Diazoacetate
[0261] Biocatalysts based on hemoglobin I from Methylacidophilum infernorum were prepared as described in Example 8 and screened for ability to catalyze the reaction shown below. See, Teh et al. (FEBS Letters 585(20):3250-8; 2011) for a description of the native protein.
##STR00047##
[0262] To carry out the cyclopropanation reaction, cell suspensions following growth and induction as in Example 8 were centrifuged using a Beckman Coulter AVANTI JXN-26 centrifuge at 4,000 rpm for 15 minutes. After discarding the supernatant, the 96-well plate was transferred to an anaerobic chamber from Coy Laboratory Products Inc. Cell pellets were resuspended in 190 l of degassed M9 minimal media using an Eppendorf MixMate Vortex Mixer at 2,000 rpm for 3 minutes. 5 L of 3,4-difluorostyrene (800 mM, in MeOH solution) was added and incubated for 5 minutes under 1200 rpm. The biocatalysis reaction was initiated by adding 5 L of ethyl diazoacetate (400 mM, in MeOH). The final concentrations of the reactants were as follows: 20 mM 3,4-difluorostyrene, 10 mM ethyl diazoacetate. The reaction was carried out using an Eppendorf MixMate Vortex Mixer at 1,500 rpm for 30 minutes.
[0263] To quench the reaction, the biocatalysis plate was taken out of anaerobic chamber, and the reaction was quenched by the addition of 350 l of hexane containing 1 mg/mL 1,3,5-trimethoxybenzene (Sigma-Aldrich) as an internal standard, and mixed using Eppendorf MixMate Vortex Mixer for 5 minutes. The 96-well plate was centrifuged in a Beckman Coulter AVANTI JXN-26 centrifuge at 4,000 rpm for 5 minutes. The organic layer was transferred into a 96-well plate (1.1 mL Axygen Scientific 96-well deep well plate, round bottom). Analysis of the product formed was carried out using an Agilent 1100 DAD/RID HPLC system, with a CHIRALCEL OJ-H column (5 um, 4.6*150 mm), isocratic elution with 5% Isopropanol in hexane containing 0.1% acetic acid.
Example 10. Identification of Variant Hemoglobins from Methylacidophilum infernorum Having Improved Stereoselectivity for the Desired Stereoisomer Trans (1R,2R)-2-(3,4-difluorophenyl)-1-cyclopropanecarboxylic Acid Ethyl Ester
[0264] Reactions were carried out in triplicate using the screening procedure described in Example 9. The results of selected variants are shown in
[0265] Where the wild type Hemoglobin I from Methylacidophilum infernorum showed a preference for producing the undesired trans isomer of 2-(3,4-difluorophenyl)-1-cyclopropanecarboxylic acid ethyl ester, several of the variants showed selectivity for producing the desired stereoisomer as the major product As an example of the improvement found, the wild type Hemoglobin I from M. infernorum produced the desired trans (1R,2R)-2-(3,4-difluorophenyl)-1-cyclopropanecarboxylic acid ethyl ester with approximately 30% ee (the negative sign indicates a 30% ee for the undesired stereoisomer) The best single variant, V95F, produced the desired trans stereoisomer with +76% ee, indicating a 75% ee for the desired stereoisomer.
Example 11. Identification of Double Variant Truncated Hemoglobins from Bacillus subtilis Having Improved Stereoselectivity for the Desired Stereoisomer Trans (1R,2R)-2-(3,4-difluorophenyl)-1-cyclopropanecarboxylic Acid Ethyl Ester
[0266] Based on analysis of the crystal structure of the truncated hemoglobin from Bacillus subtilis (GenBank Accession No. AEP90260) 2 amino acid residues were identified in the distal binding pocket for targeted mutagenesis: T45 and Q49. The following 9 targeted double mutants were generated using primer-directed mutagenesis: T45L, Q49L; T45L, Q49F; T45L, Q49A; T45F, Q49L; T45F, Q49F; T45F, Q49A; T45A, Q49L; T45A, Q49F; T45A, Q49A.
[0267] Reactions were carried out in triplicate using the screening procedure described in Example 9. The results of selected variants are shown in
Example 12. Preparation and Use of Exceptional B. Subtilis Truncated Hemoglobin Variants for Synthesis of Ticagrelor Intermediates
[0268] Library Generation and Reaction Screening in 96-Well Format.
[0269] For position Y25 of B. subtilis trHb, library was generated by employing the 22c-trick method (Kille et al. ACS Synth. Biol. 2013, 2, 83). Primers containing NDT, VHG and TGG at the desired positions were mixed in 12:9:1 ratio and then used for PCR using standard QuikChange protocol. The PCR products were gel purified, digested with DpnI, repaired using Gibson Mix, and then used to transform electrocompetent E. coli BL21(DE3) strain. E. coli libraries were cultured in a 96-well plate using LB.sub.amp (300 L/well) medium at 37 C., 220 rpm overnight. Hyperbroth.sub.amp medium (1000 L/well) was inoculated with the preculture (30 L/well), and incubated at 37 C., 220 rpm for 3 h. Concurrently, 10 out of the 96 wells were sequenced to ensure its genetic diversity at the desired position (Y25). The remaining of the preculture was used to prepare glycerol stocks of the library, which was stored at 80 C. in 96-well plate. After the indicated time, the 96-well plate expression culture was cooled on ice for 30 min, and then induced with IPTG and 5-aminolevulinic acid to final concentrations of 0.5 mM and 1.0 mM respectively. Protein expression was conducted at 20 C., 220 rpm for 24 h. The cells were pelleted (4000g, 5 min, 4 C.) and resuspended in nitrogen-free M9-N medium (1 L: 31 g Na.sub.2HPO.sub.4, 15 g KH.sub.2PO.sub.4, 2.5 g NaCl, 0.24 g MgSO.sub.4, 0.01 g CaCl.sub.2; 350 L/well). The resuspended cells were degassed in the anaerobic chamber, and 3,4-difluorostyrene, EDA, and EtOH were added to each well to final concentrations of 10 mM, 20 mM, and 5% v/v respectively. The plate reaction was shaken in the anaerobic chamber for 1 h, and then taken out to ambient atmosphere. To each well was added 1000 L of cyclohexane, and the plate was vortexed and centrifuged, and 400 L aliquots of the organic extracts were transferred to a shallow 96-well plate for analysis.
[0270] Analysis of In Vivo Reaction in 96-Well Plate.
[0271] Analytical SFC was performed with a Mettler SFC supercritical CO.sub.2 analytical chromatography system using Chiralpak AD column (4.6 mm25 cm) obtained from Daicel Chemical Industries, Ltd., eluting with 2% isopropanol in liquid CO.sub.2 as the mobile phase (2.5 mL/min flow rate). Observed retention times: 3,4-difluorostyrene-1.41 min, cis cyclopropane product-2.7-2.8 min, trans cyclopropane product (undesired enantiomer)-3.28 min, trans cyclopropane product (desired enantiomer)-4.17 min. Variants that were observed to perform better than the parent Bs trHb T45A Q49A were sequenced and re-screened in small-scale in vivo reaction as described above.
TABLE-US-00011 TABLE 10 Enantioselectivity data for Y25X site-saturation library. 1 2 3 4 5 6 7 8 9 10 11 12 A 92.4 95.8 95.7 29.1 79.4 NA 86.3 11.3 84.5 98.4 100.0 100.0 B 100.0 93.2 95.3 86.8 82.6 NA 89.4 2.1 92.9 94.4 96.1 100.0 C 100.0 95.7 58.5 43.6 100.0 NA 100.0 95.9 96.6 89.6 81.6 92.1 D 88.4 82.4 90.7 50.3 90.2 84.3 97.7 16.0 95.7 97.9 100.0 55.3 E 87.5 71.1 89.1 32.2 82.2 81.9 93.3 1.4 88.2 95.2 93.2 6.4 F 98.6 94.6 93.9 87.3 94.7 87.1 89.9 31.7 93.4 3.5 91.8 4.9 G 77.3 91.7 81.0 92.1 89.5 92.8 86.5 95.7 98.8 85.9 90.3 NA H NA 100.0 100.0 97.0 92.1 44.1 10.1 94.6 76.2 94.1 11.5 92.2 Identity of variants: B1, H2, A10 = Y25I; F1, A12, H3 = Y25L
[0272] Hit Validation of Bs10 Y25I and Bs10 Y25L.
[0273] Small-scale in vivo reactions were performed using the general procedure described in 2016 Brilinta provisional from Caltech, point 97. Deviations from the standard procedure are listed on separate columns on the table below. To investigate the viability of different E. coli strains for the biotransformation, the pET-22b vector harboring the desired gene was used to transform electrocompetent E. coli C41(DE3) and E. coli C43(DE3) (purchased from Lucigen).
TABLE-US-00012 TABLE 11 E. coli Substrate % % Protein strain OD.sub.600 concentration.sup.1 TTN trans ee 1 Bs10 BL21 30 A 5280 99 96 (DE3) 2 Bs10 BL21 30 B 2288 98 81 (DE3) 3 Bs10 C41 (DE3) 30 A 6714 99 98 4 Bs10 C41 (DE3) 30 B 5208 99 88 5 Bs10 C43 (DE3) 30 A 6033 99 97 6 Bs10 C43 (DE3) 30 B 5267 99 84 7 Bs10 Y25I BL21 30 B 4133 99 97 (DE3) 8 Bs10 Y25I C41 (DE3) 30 B 5067 99 99 9 Bs10 Y25I C43 (DE3) 30 B 6800 99 99 10 Bs10 Y25L BL21 30 B 6133 99 97 (DE3) 11 Bs10 Y25L C41 (DE3) 30 B 8667 99 98 12 Bs10 Y25L C43 (DE3) 30 B 7467 99 99 .sup.1A = 20 mM styrene, 40 mM ethyl diazoacetate; B = 50 mM styrene, 100 mM ethyl diazoacetate
[0274] Comparing % ee values for Entry 2, Entry 7, and Entry 10, it is apparent that the Bs10 Y25I variant and the Bs10 Y25L variant exhibit superior selectivity as whole-cell catalysts expressed in E. coli BL21(DE3), relative to the parent Bs10. The variants also provide comparable total activity with respect to the parent enzyme (ca. 4000-6000 total turnover number for the transformation). In addition, E. coli strains C.sub.41(DE3) and C.sub.43(DE3) are superior hosts relative to BL21(DE3) for this reaction. The C.sub.41(DE3) and C.sub.43(DE3) strains exhibit higher total turnover as shown in Entries 7-12 of Table 11, while also maintaining the enantioselectivity profile of the enzyme.
[0275] Performance of Other Globins in In Vivo Cyclopropanation of 3,4-difluorostyrene.
[0276] pET-22b vectors harboring neuroglobin (Ngb) F28V F61I H64A and Hell's Gate Globin IV (HgbIV) H71V L93A (UniprotKB accession numbers for WT proteins: Q9NPG2 and B.sub.3DVC.sub.3) were used to transform electrocompetent E. coli BL21(DE3). Performance of these proteins in in vivo cyclopropanation of 3,4-difluorostyrene was assayed following the general procedure for hemoprotein expression and small-scale in vivo reactions (2016 Brilinta provisional from Caltech, point 87, 96, and 97).
TABLE-US-00013 TABLE 12 Protein OD.sub.600 TTN % trans % ee Ngb F28V F61I H64A 30 1990 90:10 47 HgbIV H71V L93A 30 2260 93:7 41
[0277] These results show that other natural globins can be engineered to be capable catalysts for the cyclopropanation reaction. Even though initial engineered variants produced predominantly the trans diastereomer of the cyclopropane product, they made the wrong enantiomer in the reaction. However, we contend that these enzymes can be further evolved to produce the desired cyclopropane enantiomer.
Example 13. Globin-Catalyzed Cyclopropanation Reactions Using Diazoketone Reagents
[0278] Protein Purification of Truncated Hemoglobin from Bacillus Subtilis and Hemoglobin I from Methylacidophilum infernorum.
[0279] 250 mL culture of E coli BL21 DE3 (New England Biolabs) with vector pET22b carrying the gene encoding the truncated hemoglobin from Bacillus subtilis or hemoglobin I from Methylacidophilum infernorum was inoculated. The cells were grown in INNOVA shaker at 37 C. with shaking at 250 rpm until an optical density (OD.sub.600) of 1.0 was reached. The culture was then induced by adding isopropyl -D-1-thiogalactopyranoside (IPTG) and aminolevulinic acid (ALA) to a final concentration of 0.5 mM and 1 mM respectively. The culture was incubated at 28 C. with 250 rpm for 22 hours. Cell culture was spun down using Beckman Coulter AVANTI JXN-26 centrifuge at 4,000 rpm for 15 minutes. After discarding the supernatant, the cell pellets were resuspended in 25 mL of lysis buffer (50 mM KPi, 10 mM imidazole, pH 8). Sonication was conducted using Branson digital Ultrasonicator. Crude cell lysates were clarified using Beckman Coulter AVANTI JXN-26 centrifuge at 12,000 rpm for 40 minutes. Further His-tag purification was done following a known procedure (see, Bordeaux, et al. Angew. Chem. Int. Ed. 2015, 54, 1744-1748). Purified enzymes were buffer exchanged into storage buffer (50 mM KPi, pH 8). Enzyme concentration was measured as in previous examples. All variant genes were expressed and purified the same way.
[0280] Screening of Globin Variants for Cyclopropanation Activity for the Reaction of 3,4-difluorostyrene and Diazoacetone.
[0281] Diazoacetone was prepared according to a published procedure (see, Abid, et al. J. Org. Chem. 2015, 80, 9980-9988). To carry out the cyclopropanation reaction, isolated enzyme samples were transferred into an anaerobic chamber (Coy Laboratory Products Inc.). Enzymes were diluted into degassed buffer (50 mM KPi, 50 mM borate, pH 9) to a final concentration of 10 M in 1.5 mL Eppendorf tubes. Total volume was 200 L. Then, 10 L of 3,4-difluorostyrene (800 mM, in MeOH solution) was added and incubated for 5 minutes under 1200 rpm using MixMate Vortex Mixer. The biocatalysis reaction was initiated by adding 10 L of diazoacetone solution (400 mM, in MeOH). The final concentrations in the reaction mixture were as follows: 3 M enzyme, 40 mM 3,4-difluorostyrene, 20 mM diazoacetone.
##STR00048##
[0282] To quench the reaction, the biocatalysis reaction vials were taken out from the anaerobic chamber. The reaction was quenched by addition of 200 L of hexane containing 0.1% (v/v) hexadecane (Sigma-Aldrich) as internal standard, and quenched mixture was vortexed for 10 seconds using an Eppendorf MixMate Vortex Mixer. The organic layer was transferred into glass HPLC vials with inserts. Analysis of the product mixture was carried out using an Agilent 6890 GC system, Agilent Cyclosil B column (0.25 m, 0.25 mm*30 m). Chromatography was conducted using a flow rate of 1.4 mL/min and the temperature ramp shown in Table 13.
TABLE-US-00014 TABLE 13 Time (mins) Ramp ( C./min) Temperature ( C.) 0 135 2 1 165 32 20 200
[0283] Results of the hemoglobin-catalyzed cyclopropanation reactions with diazoacetone are summarized in Table 14. The GC traces included two peaks corresponding to isomeric trans products: an earlier peak at 13.1 min and a later peak at 14.3 min. The results show that the B. subtilis TrHb variants are selective for production of the earlier-eluting trans stereoisomer as the major product. The M. infernorum variant (HG I) is selective for production of the second, later-eluting trans stereoisomer as the major product. In both cases, the amount of trans product was 97% or more of the total product.
TABLE-US-00015 TABLE 14 Variant TTN Trans:cis Trans ee.sup.1 HG I Q50V/L54A 2311 99:1 68.0 BS Wildtype 0 BS T45L/Q49A 511 99:1 96.1 BS T45F/Q49A 494 97:3 96.7 BS T45A/Q49A 327 99:1 97.8 .sup.1ee calculation: ee = [(earlier trans) (later trans)]/[(earlier trans) + (later trans)]%
[0284] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.
TABLE-US-00016 INFORMALSEQUENCELISTING SEQIDNO:1 CYP102A1 CytochromeP450(BM3) Bacillusmegaterium GenBankAccessionNo.AAA87602 >gi|142798|gb|AAA87602.1| cytochromeP-450:NADPH-P-450reductaseprecursor [Bacillusmegaterium] TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIK EACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMM VDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVR ALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLN GKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLV DPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQ LHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHN TPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGH PPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAD RGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMH GAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFG LDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVE LEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLI MVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIIT LHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYAD VHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:2 CYP102A1 B.megaterium >gi|281191140|gb|ADA57069.1| NADPH-cytochromeP450reductase102A1V9 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVATREGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:3 CYP102A1 B.megaterium >gi|281191138|gb|ADA57068.1| NADPH-cytochromeP450reductase102A1V10 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETFAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVATRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:4 CYP102A1 B.megaterium >gi|281191126|gb|ADA57062.1| NADPH-cytochromeP450reductase102A1V4 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFN LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSERSTRHLEIALPKEASYQEGDH LGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSIRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP FRSFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:5 CYP102A1 B.megaterium >gi|281191124|gb|ADA57061.1| NADPH-cytochromeP450reductase102A1V8 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPRVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVATRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:6 CYP102A1 B.megaterium >gi|281191120|gb|ADA57059.1| NADPH-cytochromeP450reductase102A1V3 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFN LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDH LGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM ERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:7 CYP102A1 B.megaterium >gi|281191118|gb|ADA57058.1| NADPH-cytochromeP450reductase102A1V7 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPPEGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVATRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNEPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:8 CYP102A1 B.megaterium >gi|281191112|gb|ADA57055.1|NADPH-cytochromeP450reductase102A1V2 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFN LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDH LGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM ERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:9 CYP102A1 B.megaterium >gi|269315992|gb|ACZ37122.1|cytochromeP450:NADPHP450reductase[Bacillus megaterium] MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVATRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:10 CYP102A1 B.megaterium >gi|281191116|gb|ADA57057.1|NADPH-cytochromeP450reductase102A1V6 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVTTRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVV EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAG SEQIDNO:11 CYP102A1 B.megaterium >gi|281191114|gb|ADA57056.1|NADPH-cytochromeP450reductase102A1V5 [Bacillusmegaterium] MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHI EVPEDMTRLTLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDI KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEV KGVRYSVFGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN LNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDH LGVIPRNYEGIVNRVTTRFGLDASQQIRLEAEEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA KTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAP FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVV EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAG SEQIDNO:12 CYP153A6 Mycobacteriumsp.EXN-1500 GenBankAccessionNo.:CAH04396 >gi|519971171embICAH04396.1|cytochromeP450alkanehydroxylase [Mycobacteriumsp.HXN-1500] 1MTEMTVAASDATNAAYGMALEDIDVSNPVLFRDNTWHPYFKRLREEDPVHYCKSSMFGPY 61WSVTKYRDIMAVETNPKVFSSEAKSGGITIMDDNAAASLPMFIAMDPPKHDVQRKTVSPI 121VAPENLATMESVIRQRTADLLDGLPINEEFDWVHRVSIELTTKMLATLFDFPWDDRAKLT 181RWSDVTTALPGGGIIDSEEQRMAELMECATYFTELWNQRVNAEPKNDLISMMAHSESTRH 241MAPEEYLGNIVLLIVGGNDTTRNSMTGGVLALNEFPDEYRKLSANPALISSMVSEIIRWQ 301TPLSHMRRTALEDIEFGGKHIRQGDKVVMWYVSGNRDPEAIDNPDTFIIDRAKPRQHLSF 361GFGIHRCVGNRLAELQLNILWEEILKRWPDPLQIQVLQEPTRVLSPFVKGYESLPVRINA SEQIDNO:13 CYP5013C2 Tetrahymenathermophile GenBankAccessionNo.:ABY59989 >gi|164519863|gb|ABY59989.1|cytochromeP450monooxygenaseCYP5013C2 [Tetrahymenathermophila] 1MIFELILIAVALFAYFKIAKPYFSYLKYRKYGKGFYYPILGEMIEQEQDLKQHADADYSV 61HHALDKDPDQKLFVTNLGTKVKLRLIEPEIIKDFFSKSQYYQKDQTFIQNITRFLKNGIV 121FSEGNTWKESRKLFSPAFHYEYIQKLTPLINDITDTIFNLAVKNQELKNFDPIAQIQEIT 181GRVIIASFFGEVIEGEKFQGLTIIQCLSHIINTLGNQTYSIMYFLFGSKYFELGVTEEHR 241KFNKFIAEFNKYLLQKIDQQIEIMSNELQTKGYIQNPCILAQLISTHKIDEITRNQLFQD 301FKTFYIAGMDTTGHLLGMTIYYVSQNKDIYTKLQSEIDSNTDQSAHGLIKNLPYLNAVIK 361ETLRYYGPGNILFDRIAIKDHELAGIPIKKGTIVTPYAMSMQRNSKYYQDPHKYNPSRWL 421EKQSSDLHPDANIPFSAGQRKCIGEQLALLEARIILNKFIKMFDFTCPQDYKLMMNYKFL 481SEPVNPLPLQLTLRKQ SEQIDNO:14 Nonomuraeadietziae >gi|445067389|gb|AGE14547.1|cytochromeP450hydroxylasesb8[Nonomuraea dietziae] GenBankAccessionNo.:AGE14547 VNIDLVDQDHYATEGPPHEQMRWLREHAPVYWHEGEPGFWAVTRHEDVVHVSRHSDLESSARRLALFNEMPEEQR ELQRMMMLNQDPPEHTRRRSLVNRGETPRTIRALEQHIRDICDDLLDQCSGEGDFVTDLAAPLPLYVICELLGAP VADRDKIFAWSNRMIGAQDPDYAASPEEGGAAAMEVYAYASELAAQRRAAPRDDIVTKLLQSDENGESLTENEFE LFVLLLVVAGNETTRNAASGGMLTLFEHPDQWDRLVADPSLAATAADEIVRWVSPVNLFRRTATADLTLGGQQVK ADDKVVVEYSSANRDASVESDPEVEDIGRSPNPHIGEGGGGAHFCLGNHLAKLELRVLFEQLARREPRMRQTGEA RRLRSNFINGIKTLPVTLG SEQIDNO:15 CYP2R1 Homosapiens GenBankAccessionNo.:NP078790 >gi|45267826|ref|NP_078790.21vitaminD25-hydroxylase[Homosapiens] 1MWKLWRAEEGAAALGGALFLLLFALGVRQLLKQRRPMGFPPGPPGLPFIGNIYSLAASSE 61LPHVYMRKQSQVYGEIFSLDLGGISTVVLNGYDVVKECLVHQSEIFADRPCLPLFMKMTK 121MGGLLNSRYGRGWVDHRRLAVNSFRYFGYGQKSFESKILEETKFFNDAIETYKGRPFDFK 181QLITNAVSNITNLIIFGERFTYEDTDFQHMIELFSENVELAASASVFLYNAFPWIGILPF 241GKHQQLFRNAAVVYDFLSRLIEKASVNRKPQLPQHFVDAYLDEMDQGKNDPSSTFSKENL 301IFSVGELIIAGTETTTNVLRWAILFMALYPNIQGQVQKEIDLIMGPNGKPSWDDKCKMPY 361TEAVLHEVLRFCNIVPLGIFHATSEDAVVRGYSIPKGTTVITNLYSVHFDEKYWRDPEVF 421HPERFLDSSGYFAKKEALVPFSLGRRHCLGEHLARMEMFLFFTALLQRFHLHFPHELVPD 481LKPRLGMTLQPQPYLICAERR SEQIDNO:16 CYP2R1 Maccamulatta GenBankAccessionNo.:NP001180887 >gi|302565346|ref|NP_001180887.1|vitaminD25-hydroxylase[Maccamulatta] 1MWKLWGGEEGAAALGGALFLLLFALGVRQLLKLRRPMGFPPGPPGLPFIGNIYSLAASAE 61LPHVYMRKQSQVYGEIFSLDLGGISTVVLNGYDVVKECLVHQSGIFADRPCLPLFMKMTK 121MGGLLNSRYGQGWVEHRRLAVNSFRYFGYGQKSFESKILEETKFFTDAIETYKGRPFDFK 181QLITSAVSNITNLIIFGERFTYEDTDFQHMIELFSENVELAASASVFLYNAFPWIGILPF 241GKHQQLFRNASVVYDFLSRLIEKASVNRKPQLPQHFVDAYFDEMDQGKNDPSSTFSKENL 301IFSVGELIIAGTETTTNVLRWAILFMALYPNIQGQVQKEIDLIMGPNGKPSWDDKFKMPY 361TEAVLHEVLRFCNIVPLGIFHATSEDAVVRGYSIPKGTTVITNLYSVHFDEKYWRDPEVF 421HPERFLDSSGYFAKKEALVPFSLGRRHCLGEQLARMEMFLFFTALLQRFHLHFPHELVPD 481LKPRLGMTLQPQPYLICAERR SEQIDNO:17 CYP2R1 Canisfamiliaris GenBankAccessionNo.:XP_854533 >gi|73988871|ref|XP_854533.1|PREDICTED:vitaminD25-hydroxylase[Canis lupusfamiliaris] 1MRGPPGAEACAAGLGAALLLLLFVLGVRQLLKQRRPAGFPPGPSGLPFIGNIYSLAASGE 61LAHVYMRKQSRVYGEIFSLDLGGISAVVLNGYDVVKECLVHQSEIFADRPCLPLFMKMTK 121MGGLLNSRYGRGWVDHRKLAVNSFRCFGYGQKSFESKILEETNFFIDAIETYKGRPFDLK 181QLITNAVSNITNLIIFGERFTYEDTDFQHMIELFSENVELAASASVFLYNAFPWIGIIPF 241GKHQQLFRNAAVVYDFLSRLIEKASINRKPQSPQHFVDAYLNEMDQGKNDPSCTFSKENL 301IFSVGELIIAGTETTTNVLRWAILFMALYPNIQGQVQKEIDLIMGPTGKPSWDDKCKMPY 361TEAVLHEVLRFCNIVPLGIFHATSEDAVVRGYSIPKGTTVITNLYSVHFDEKYWRNPEIF 421YPERFLDSSGYFAKKEALVPFSLGKRHCLGEQLARMEMFLFFTALLQRFHLHFPHGLVPD 481LKPRLGMTLQPQPYLICAERR SEQIDNO:18 CYP2R1 Musmusculus GenBankAccessionNo.:AAI08963 >gi|80477959|gb|AAI08963.1|Cyp2r1protein[Musmusculus] 1MGDEMDQGQNDPLSTFSKENLIFSVGELIIAGTETTTNVLRWAILFMALYPNIQGQVHKE 61IDLIVGHNRRPSWEYKCKMPYTEAVLHEVLRFCNIVPLGIFHATSEDAVVRGYSIPKGTT 121VITNLYSVHFDEKYWKDPDMFYPERFLDSNGYFTKKEALIPFSLGRRHCLGEQLARMEMF 181LFFTSLLQQFHLHFPHELVPNLKPRLGMTLQPQPYLICAERR SEQIDNO:19 CYP152A6 BacillushaloduransC-125 GenBankAccessionNo.:NP_242623 >gi|15614320|ref|NP_242623.1|fattyacidalphahydroxylase[Bacillus haloduransC-125] 1MKSNDPIPKDSPLDHTMNLMREGYEFLSHRMERFQTDLFETRVMGQKVLCIRGAEAVKLF 61YDPERFKRHRATPKRIQKSLFGENAIQTMDDKAHLHRKQLFLSMMKPEDEQELARLTHET 121WRRVAEGWKKSRPIVLFDEAKRVLCQVACEWAEVPLKSTEIDRRAEDFHAMVDAFGAVGP 181RHWRGRKGRRRTERWIQSIIHQVRTGSLQAREGSPLYKVSYHRELNGKLLDERMAAIELI 241NVLRPIVAIATFISFAAIALQEHPEWQERLKNGSNEEFHMFVQEVRRYYPFAPLIGAKVR 301KSFTWKGVRFKKGRLVFLDMYGTNHDPKLWDEPDAFRPERFQERKDSLYDFIPQGGGDPT 361KGHRCPGEGITVEVMKTTMDFLVNDIDYDVPDQDISYSLSRMPTRPESGYIMANIERKYE 421HA SEQIDNO:20 aryC Streptomycesparvus GenBankAccessionNo.:AFM80022 >gi|392601346|gb|AFM80022.1|cytochromeP450[Streptomycesparvus] 1MYLGGRRGTEAVGESREPGVWEVFRYDEAVQVLGDHRTFSSDMNHFIPEEQRQLARAARG 61NFVGIDPPDHTQLRGLVSQAFSPRVTAALEPRIGRLAEQLLDDIVAERGDKASCDLVGEF 121AGPLSAIVIAELFGIPESDHTMIAEWAKALLGSRPAGELSIADEAAMQNTADLVRRAGEY 181LVHHITERRARPQDDLTSRLATTEVDGKRLDDEEIVGVIGMFLIAGYLPASVLTANTVMA 241LDEHPAALAEVRSDPALLPGAIEEVLRWRPPLVRDQRLTTRDADLGGRTVPAGSMVCVWL 301ASAHRDPFRFENPDLFDIHRNAGRHLAFGKGIHYCLGAPLARLEARIAVETLLRRFERIE 361IPRDESVEFHESIGVLGPVRLPTTLFARR SEQIDNO:21 CYP101A1 Pseudomonasputida UniprotAccessionNo.:P00183 >sp1P001831CPXA_PSEPUCamphor5-monooxygenaseOS= Pseudomonasputida GN= camCPE= 1SV= 2 TTETIQSNANLAPLPPHVPEHLVEDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAY EDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNF TEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVAN GQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSLVADGRI LTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWL TRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV SEQIDNO:22 Homosapiens CYP2D7 GenBankAccessionNo.:AA049806 >gi|37901459|gb|AA049806.1|cytochromeP450[Homosapiens] GLEALVPLAMIVAIFLLLVDLMHRHQRWAARYPPGPLPLPGLGNLLHVDFQNTPYCFDQ LRRRFGDVFNLQLAWTPVVVLNGLAAVREAMVTRGEDTADRPPAPIYQVLGFGPRSQGVI LSRYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEAACLCAAFADQAGRPFRPNGLLDK AVSNVIASLTCGRRFEYDDPRFLRLLDLAQEGLKEESGFLREVLNAVPVLPHIPALAGKV LRFQKAFLTQLDELLTEHRMTWDPAQPPRDLTEAFLAKKEKAKGSPESSFNDENLRIVVG NLFLAGMVTTLTTLAWGLLLMILHLDVQRGRRVSPGCSPIVGTHVCPVRVQQEIDDVIGQ VRRPEMGDQVHMPYTTAVIHEVQRFGDIVPLGVTHMTSRDIEVQGFRIPKGTTLITNLSS VLKDEAVWEKPFRFHPEHFLDAQGHFVKPEAFLPFSAGRRACLGEPLARMELFLFFTSLL QHFSFSVAAGQPRPSHSRVVSFLVTPSPYELCAVPR SEQIDNO:23 Rattusnorvegicus CYPC27 GenBankAccessionNo.:AAB02287 >gi|1374714|gb|AAB02287.1|cytochromeP450[Rattusnorvegicus] AVLSRMRLRWALLDTRVMGHGLCPQGARAKAAIPAALRDHESTEGPGTGQDRPRLRSLAELPGPGTLRF LFQLFLRGYVLHLHELQALNKAKYGPMWTTTEGTRTNVNLASAPLLEQVMRQEGKYPIRDSMEQWKEHRD HKGLSYGIFITQGQQWYHLRHSLNQRMLKPAEAALYTDALNEVISDFIARLDQVRTESASGDQVPDVAHL LYHLALEAICYILFEKRVGCLEPSIPEDTATFIRSVGLMFKNSVYVTFLPKWSRPLLPFWKRYMNNWDNI FSFGEKMIHQKVQEIEAQLQAAGPDGVQVSGYLHFLLTKELLSPQETVGTFPELILAGVDTTSNTLTWAL YHLSKNPEIQEALHKEVTGVVPFGKVPQNKDFAHMPLLKAVIKETLRLYPVVPTNSRIITEKETEINGFL FPKNTQFVLCTYVVSRDPSVFPEPESFQPHRWLRKREDDNSGIQHPFGSVPFGYGVRSCLGRRIAELEMQ LLLSRLIQKYEVVLSPGMGEVKSVSRIVLVPSKKVSLRFLQRQ SEQIDNO:24 CYP2B4 Oryctolaguscuniculus GenBankAccessionNo.AAA65840 >gi|164959|gb|AAA65840.1|cytochromeP-450[Oryctolaguscuniculus] MEFSLLLLLAFLAGLLLLLFRGHPKAHGRLPPGPSPLPVLGNLLQMDRKGLLRSFLRLRE KYGDVFTVYLGSRPVVVLCGTDAIREALVDQAEAFSGRGKIAVVDPIFQGYGVIFANGER WRALRRFSLATMRDFGMGKRSVEERIQEEARCLVEELRKSKGALLDNTLLFHSITSNIIC SIVEGKREDYKDPVFLRLLDLFFQSFSLISSFSSQVFELFPGFLKHFPGTHRQTYRNLQE INTFIGQSVEKHRATLDPSNPRDFIDVYLLRMEKDKSDPSSEFHHQNLILTVLSLFFAGT ETTSTTLRYGELLMLKYPHVTERVQKEIEQVIGSHRPPALDDRAKMPYTDAVIHEIQRLG DLIPFGVPHTVTKDTQFRGYVIPKNTEVFPVLSSALHDPRYFETPNTFNPGHFLDANGAL KRNEGFMPFSLGKRICLGEGIARTELFLFFTTILQNFSIASPVPPEDIDLTPRESGVGNV PPSYQIRFLAR SEQIDNO:25 CYP102A2 Bacillussubtilis UniprotAccessionNo.008394 >sp10083941CYPD_BACSUProbablebifunctionalP-450/NADPH-P450reductase1 OS= Bacillussubtilis(strain168)GN= cypDPE= 1SV= 2 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELV KEVCDEERFDKSIEGALEKVRAFSGDGLFTSWTHEPNWRKAHNILMPTFSQRAMKDYHEK MVDIAVQLIQKWARLNPNEAVDVPGDMTRLTLDTIGLCGENYRENSYYRETPHPFINSMV RALDEAMHQMQRLDVQDKLMVRTKRQFRHDIQTMESLVDSIIAERRANGDQDEKDLLARM LNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRV LTDAAPTYKQVLELTYIRMILNESLRLWPTAPAFSLYPKEDTVIGGKFPITTNDRISVLI PQLHRDRDAWGKDAEEFRPERFEHQDQVPHHAYKPFGNGQRACIGMQFALHEATLVLGMI LKYFTLIDHENYELDIKQTLTLKPGDFHIRVQSRNQDAIHADVQAVEKAASDEQKEKTEA KGTSVIGLNNRPLLVLYGSDTGTAEGVARELADTASLHGVRTETAPLNDRIGKLPKEGAV VIVTSSYNGKPPSNAGQFVQWLQEIKPGELEGVHYAVFGCGDHNWASTYQYVPRFIDEQL AEKGATRFSARGEGDVSGDFEGQLDEWKKSMWADAIKAFGLELNENADKERSTLSLQFVR GLGESPLARSYEASHASIAENRELQSADSDRSTRHIEIALPPDVEYQEGDHLGVLPKNSQ TNVSRILHRFGLKGTDQVTLSASGRSAGHLPLGRPVSLHDLLSYSVEVQEAATRAQIREL AAFTVCPPHRRELEELSAEGVYQEQILKKRISMLDLLEKYEACDMPFERFLELLRPLKPR YYSISSSPRVNPRQASITVGVVRGPAWSGRGEYRGVASNDLAERQAGDDVVMFIRTPESR FQLPKDPETPIIMVGPGTGVAPFRGFLQARDVLKREGKTLGEAHLYFGCRNDRDFIYRDE LERFEKDGIVTVHTAFSRKEGMPKTYVQHLMADQADTLISILDRGGRLYVCGDGSKMAPD VEAALQKAYQAVHGTGEQEAQNWLRHLQDTGMYAKDVWAGI SEQIDNO:26 CYP102A3 Bacillussubtilis UniprotAccessionNo.008336 >sp10083361CYPE_BACSUProbablebifunctionalP-450/NADPH-P450reductase2 OS= Bacillussubtilis(strain168)GN= cypEPE= 1SV= 2 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFRFDFPGVSSVFVSGHNLV AEVCDESRFDKNLGKGLQKVREFGGDGLFTSWTHEPNWQKAHRILLPSFSQKAMKGYHSM MLDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDSQHPFITSML RALKEAMNQSKRLGLQDKMMVKTKLQFQKDIEVMNSLVDRMIAERKANPDDNIKDLLSLM LYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRV LTDDTPEYKQIQQLKYTRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLI PKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLV LKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQADIKAETKPKETKP KHGTPLLVLYGSNLGTAEGIAGELAAQGRQMGETAETAPLDDYIGKLPEEGAVVIVTASY NGSPPDNAAGFVEWLKELEEGQLKGVSYAVFGCGNRSWASTYQRIPRLIDDMMKAKGASR LTEIGEGDAADDFESHRESWENRFWKETMDAFDINEIAQKEDRPSLSIAFLSEATETPVA KAYGAFEGVVLENRELQTADSTRSTRHIELEIPAGKTYKEGDHIGIMPKNSRELVQRVLS RFGLQSNHVIKVSGSAHMSHLPMDRPIKVADLLSSYVELQEPASRLQLRELASYTVCPPH QKELEQLVLDDGIYKEQVLAKRLTMLDFLEDYPACEMPFERFLALLPSLKPRYYSISSSP KVHANIVSMTVGVVKASAWSGRGEYRGVASNYLAELNTGDAAACFIRTPQSGFQMPDEPE TPMIMVGPGTGIAPFRGFIQARSVLKKEGSTLGEALLYFGCRRPDHDDLYREELDQAEQE GLVTIRRCYSRVENESKGYVQHLLKQDSQKLMTLIEKGAHIYVCGDGSQMAPDVEKTLRW AYETEKGASQEESADWLQKLQDQKRYIKDVWTGN SEQIDNO:27 CYP102A1 B.megateriumDSM32 UniprotAccessionNo.P14779 >sp1P147791CPXB_BACMEBifunctionalP-450/NADPH-P450reductaseOS= Bacillus megateriumGN= cyp102A1PE= 1SV= 2 1MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIK 61EACDESRFDKNLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMM 121VDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVR 181ALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKASGEQSDDLLTHMLN 241GKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLV 301DPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQ 361LHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 421HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHN 481TPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGH 541PPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIAD 601RGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMH 661GAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFG 721LDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVE 781LEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDE 841KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLI 901MVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIIT 961LHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYAD 1021VHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:28 CYP102A5 B.cereusATCC14579 GenBankAccessionNo.AAP10153 >gi|29896875|gb|AAP10153.1|NADPH-cytochromeP450reductase[Bacillus cereusATCC145791 1MEKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKIAEEYGPIFQIQTLSDTIIVVSGHEL 61VAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETHEPNWKKAHNILMPTFSQRAMKDYHA 121MMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSM 181TRALDEAMHQLQRLDIEDKLMWRTKRQFQHDIQSMFSLVDNIIAERKSSGDQEENDLLSR 241MLNVPDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDR 301VLTDPTPTYQQVMKLKYMRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISV 361LIPQLHRDKDAWGDNVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFALHEATLVMG 421MLLQHFELIDYQNYQLDVKQTLTLKPGDFKIRILPRKQTISHPTVLAPTEDKLKNDEIKQ 481HVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVVALNDRIGSLPKE 541GAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYID 601EQMAQKGATRFSKRGEADASGDFEEQLEQWKQNMWSDAMKAFGLELNKNMEKERSTLSLQ 661FVSRLGGSPLARTYEAVYASILENRELQSSSSDRSTRHIEVSLPEGATYKEGDHLGVLPV 721NSEKNINRILKRFGLNGKDQVILSASGRSINHIPLDSPVSLLALLSYSVEVQEAATRAQI 781REMVTFTACPPHKKELEALLEEGVYHEQILKKRISMLDLLEKYEACEIRFERFLELLPAL 841KPRYYSISSSPLVAHNRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTP 901QSNFELPKDPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMNLGQAHLYFGCRHPEKDYL 961YRTELENDERDGLISLHTAFSRLEGHPKTYVQHLIKQDRINLISLLDNGAHLYICGDGSK 1021MAPDVEDTLCQAYQEIHEVSEQEARNWLDRVQDEGRYGKDVWAGI SEQIDNO:29 CYP102A7 B.licheniformisATTC1458 GenBankAccessionNo.YP079990 >gi|520811991reflYP_079990.1|cytochromeP450/NADPH-ferrihemoprotein reductase[BacilluslicheniformisDSM13=ATCC145801 1MNKLDGIPIPKTYGPLGNLPLLDKNRVSQSLWKIADEMGPIFQFKFADAIGVFVSSHELV 61KEVSEESRFDKNMGKGLLKVREFSGDGLFTSWTEEPNWRKAHNILLPSFSQKAMKGYHPM 121MQDIAVQLIQKWSRLNQDESIDVPDDMTRLTLDTIGLCGFNYRFNSFYREGQHPFIESMV 181RGLSEAMRQTKRFPLQDKLMIQTKRRFNSDVESMFSLVDRIIADRKQAESESGNDLLSLM 241LHAKDPETGEKLDDENIRYQIITFLIAGHETTSGLLSFAIYLLLKHPDKLKKAYEEADRV 301LTDPVPSYKQVQQLKYIRMILNESIRLWPTAPAFSLYAKEETVIGGKYLIPKGQSVTVLI 361PKLHRDQSVWGEDAEAFRPERFEQMDSIPAHAYKPFGNGQRACIGMQFALHEATLVLGMI 421LQYFDLEDHANYQLKIKESLTLKPDGFTIRVRPRKKEAMTAMPGAQPEENGRQEERPSAP 481AAENTHGTPLLVLYGSNLGTAEEIAKELAEEAREQGFHSRTAELDQYAGAIPAEGAVIIV 541TASYNGNPPDCAKEFVNWLEHDQTDDLRGVKYAVFGCGNRSWASTYQRIPRLIDSVLEKK 601GAQRLHKLGEGDAGDDFEGQFESWKYDLWPLLRTEFSLAEPEPNQTETDRQALSVEFVNA 661PAASPLAKAYQVFTAKISANRELQCEKSGRSTRHIEISLPEGAAYQEGDHLGVLPQNSEV 721LIGRVFQRFGLNGNEQILISGRNQASHLPLERPVHVKDLFQHCVELQEPATRAQIRELAA 781HTVCPPHQRELEDLLKDDVYKDQVLNKRLTMLDLLEQYPACELPFARFLALLPPLKPRYY 841SISSSPQLNPRQTSITVSVVSGPALSGRGHYKGVASNYLAGLEPGDAISCFIREPQSGFR 901LPEDPETPVIMVGPGTGIAPYRGFLQARRIQRDAGVKLGEAHLYFGCRRPNEDFLYRDEL 961EQAEKDGIVHLHTAFSRLEGRPKTYVQDLLREDAALLIHLLNEGGRLYVCGDGSRMAPAV 1021EQALCEAYRIVQGASREESQSWLSALLEEGRYAKDVWDGGVSQHNVKADCIART SEQIDNO:30 CYPX B.thuringiensisserovarkonkukian str.97-27 GenBankAccessionNo.YP037304 >gi|494800991reflYP_037304.1|NADPH-cytochromeP450reductase[Bacillus thuringiensisserovarkonkukianstr.97-271 1MDKKVSAIPQPKTYGPLGNLPLIDKDKPTLSFIKLAEEYGPIFQIQTLSDTIIVVSGHEL 61VAEVCDETRFDKSIEGALAKVRAFAGDGLFTSETDEPNWKKAHNILMPTFSQRAMKDYHA 121MMVDIAVQLVQKWARLNPNENVDVPEDMTRLTLDTIGLCGFNYRFNSFYRETPHPFITSM 181TRALDEAMHQLQRLDIEDKLMWRTKRQFQHDIQSMFSLVDNIIAERKSSENQEENDLLSR 241MLNVQDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYFLLKNPDKLKKAYEEVDR 301VLTDSTPTYQQVMKLKYIRMILNESLRLWPTAPAFSLYAKEDTVIGGKYPIKKGEDRISV 361LIPQLHRDKDAWGDDVEEFQPERFEELDKVPHHAYKPFGNGQRACIGMQFALHEATLVMG 421MLLQHFEFIDYEDYQLDVKQTLTLKPGDFKIRIVPRNQTISHTTVLAPTEEKLKKHEIKK 481QVQKTPSIIGADNLSLLVLYGSDTGVAEGIARELADTASLEGVQTEVVALNDRIGSLPKE 541GAVLIVTSSYNGKPPSNAGQFVQWLEELKPDELKGVQYAVFGCGDHNWASTYQRIPRYID 601EQMAQKGATRFSTRGEADASGDFEEQLEQWKQSMWSDAMKAFGLELNKNMEKERSTLSLQ 661FVSRLGGSPLARTYEAVYASILENRELQSSSSERSTRHIEISLPEGATYKEGDHLGVLPI 721NNEKNVNRILKRFGLNGKDQVILSASGRSVNHIPLDSPVRLYDLLSYSVEVQEAATRAQI 781REMVTFTACPPHKKELESLLEDGVYQEQILKKRISMLDLLEKYEACEIRFERFLELLPAL 841KPRYYSISSSPLVAQDRLSITVGVVNAPAWSGEGTYEGVASNYLAQRHNKDEIICFIRTP 901QSNFQLPENPETPIIMVGPGTGIAPFRGFLQARRVQKQKGMKVGEAHLYFGCRHPEKDYL 961YRTELENDERDGLISLHTAFSRLEGHPKTYVQHVIKEDRIHLISLLDNGAHLYICGDGSK 1021MAPDVEDTLCQAYQEIHEVSEQEARNWLDRLQEEGRYGKDVWAGI SEQIDNO:31 CYP102E1 R.metalliduransCH34 GenBankAccessionNo.YP585608 >gi|943123981reflYP_585608.1|putativebifunctionalP-450:NADPH-P450 reductase2[CupriavidusmetalliduransCH34] 1MSTATPAAALEPIPRDPGWPIFGNLFQITPGEVGQHLLARSRHHDGIFELDFAGKRVPFV 61SSVALASELCDATRFRKIIGPPLSYLRDMAGDGLFTAHSDEPNWGCAHRILMPAFSQRAM 121KAYFDVMLRVANRLVDKWDRQGPDADIAVADDMTRLTLDTIALAGFGYDFASFASDELDP 181FVMAMVGALGEAMQKLTRLPIQDRFMGRAHRQAAEDIAYMRNLVDDVIRQRRVSPTSGMD 241LLNLMLEARDPETDRRLDDANIRNQVITFLIAGHETTSGLLTFALYELLRNPGVLAQAYA 301EVDTVLPGDALPVYADLARMPVLDRVLKETLRLWPTAPAFAVAPFDDVVLGGRYRLRKDR 361RISVVLTALHRDPKVWANPERFDIDRFLPENEAKLPAHAYMPFGQGERACIGRQFALTEA 421KLALALMLRNFAFQDPHDYQFRLKETLTIKPDQFVLRVRRRRPHERFVTRQASQAVADAA 481QTDVRGHGQAMTVLCASSLGTARELAEQIHAGAIAAGFDAKLADLDDAVGVLPTSGLVVV 541VAATYNGRAPDSARKFEAMLDADDASGYRANGMRLALLGCGNSQWATYQAFPRRVFDFFI 601TAGAVPLLPRGEADGNGDFDQAAERWLAQLWQALQADGAGTGGLGVDVQVRSMAAIRAET 661LPAGTQAFTVLSNDELVGDPSGLWDFSIEAPRTSTRDIRLQLPPGITYRTGDHIAVWPQN 721DAQLVSELCERLDLDPDAQATISAPHGMGRGLPIDQALPVRQLLTHFIELQDVVSRQTLR 781ALAQATRCPFTKQSIEQLASDDAEHGYATKVVARRLGILDVLVEHPAIALTLQELLACTV 841PMRPRLYSIASSPLVSPDVATLLVGTVCAPALSGRGQFRGVASTWLQHLPPGARVSASIR 901TPNPPFAPDPDPAAPMLLIGPGTGIAPFRGFLEERALRKMAGNAVTPAQLYFGCRHPQHD 961WLYREDIERWAGQGVVEVHPAYSVVPDAPRYVQDLLWQRREQVWAQVRDGATIYVCGDGR 1021RMAPAVRQTLIEIGMAQGGMTDKAASDWFGGLVAQGRYRQDVFN SEQIDNO:32 CYP505X A.fumigatusAf293 GenBankAccessionNo.EAL92660 >gi|66852335|gb|EAL92660.1|P450familyfattyacidhydroxylase,putative [AspergillusfumigatusAf293] 1MSESKTVPIPGPRGVPLLGNIYDIEQEVPLRSINLMADQYGPIYRLTTFGWSRVFVSTHE 61LVDEVCDEERFTKVVTAGLNQIRNGVHDGLFTANFPGEENWAIAHRVLVPAFGPLSIRGM 121FDEMYDIATQLVMKWARHGPTVPIMVTDDFTRLTLDTIALCAMGTRFNSFYHEEMHPFVE 181AMVGLLQGSGDRARRPALLNNLPTSENSKYWDDIAFLRNLAQELVEARRKNPEDKKDLLN 241ALILGRDPKTGKGLTDESIIDNMITFLIAGHETTSGLLSFLFYYLLKTPNAYKKAQEEVD 301SVVGRRKITVEDMSRLPYLNAVMRETLRLRSTAPLIAVHAHPEKNKEDPVTLGGGKYVLN 361KDEPIVIILDKLHRDPQVYGPDAEEFKPERMLDENFEKLPKNAWKPFGNGMRACIGRPFA 421WQEALLVVAILLQNFNFQMDDPSYNLHIKQTLTIKPKDFHMRATLRHGLDATKLGIALSG 481SADRAPPESSGAASRVRKQATPPAGQLKPMHIFFGSNTGTCETFARRLADDAVGYGFAAD 541VQSLDSAMQNVPKDEPVVFITASYEGQPPDNAAHFFEWLSALKENELEGVNYAVFGCGHH 601DWQATFHRIPKAVNQLVAEHGGNRLCDLGLADAANSDMFTDFDSWGESTFWPAITSKFGG 661GKSDEPKPSSSLQVEVSTGMRASTLGLQLQEGLVIDNQLLSAPDVPAKRMIRFKLPSDMS 721YRCGDYLAVLPVNPTSVVRRAIRRFDLPWDAMLTIRKPSQAPKGSTSIPLDTPISAFELL 781STYVELSQPASKRDLTALADAAITDADAQAELRYLASSPTRFTEEIVKKRMSPLDLLIRY 841PSIKLPVGDFLAMLPPMRVRQYSISSSPLADPSECSITFSVLNAPALAAASLPPAERAEA 901EQYMGVASTYLSELKPGERAHIAVRPSHSGFKPPMDLKAPMIMACAGSGLAPFRGFIMDR 961AEKIRGRRSSVGADGQLPEVEQPAKAILYVGCRTKGKDDIHATELAEWAQLGAVDVRWAY 1021SRPEDGSKGRHVQDLMLEDREELVSLFDQGARIYVCGSTGVGNGVRQACKDIYLERRRQL 1081RQAARERGEEVPAEEDEDAAAEQFLDNLRTKERYATDVFT SEQIDNO:33 CYP505A8 A.nidulansFGSCA4 GenBankAccessionNo.EAA58234 >gi|40739044|gb|EAA58234.1|hypotheticalproteinAN6835.2[Aspergillus nidulansFGSCA4] 1MAEIPEPKGLPLIGNIGTIDQEFPLGSMVALAEEHGEIYRLRFPGRTVVVVSTHALVNET 61CDEKRFRKSVNSALAHVREGVHDGLFTAKMGEVNWEIAHRVLMPAFGPLSIRGMFDEMHD 121IASQLALKWARYGPDCPIMVTDDFTRLTLDTLALCSMGYRFNSYYSPVLHPFIEAMGDFL 181TEAGEKPRRPPLPAVFFRNRDQKFQDDIAVLRDTAQGVLQARKEGKSDRNDLLSAMLRGV 241DSQTGQKMTDESIMDNLITFLIAGHETTSGLLSFVFYQLLKHPETYRTAQQEVDNVVGQG 301VIEVSHLSKLPYINSVLRETLRLNATIPLFTVEAFEDTLLAGKYPVKAGETIVNLLAKSH 361LDPEVYGEDALEFKPERMSDELFNARLKQFPSAWKPFGNGMRACIGRPFAWQEALLVMAM 421LLQNFDFSLADPNYDLKFKQTLTIKPKDMFMKARLRHGLTPTTLERRLAGLAVESATQDK 481IVTNPADNSVTGTRLTILYGSNSGTCETLARRIAADAPSKGFHVMRFDGLDSGRSALPTD 541HPVVIVTSSYEGQPPENAKQFVSWLEELEQQNESLQLKGVDFAVFGCFKEWAQTFHRIPK 601LVDSLLEKLGGSRLTDLGLADVSTDELFSTFETWADDVLWPRLVAQYGADGKTQAHGSSA 661GHEAASNAAVEVTVSNSRTQALRQDVGQAMVVETRLLTAESEKERRKKHLEIRLPDGVSY 721TAGDYLAVLPINPPETVRRAMRQFKLSWDAQITIAPSGPTTALPTDGPIAANDIFSTYVE 781LSQPATRKDLRIMADATTDPDVQKILRTYANETYTAEILTKSISVLDILEQHPAIDLPLG 841TFLLMLPSMRMRQYSISSSPLLTPTTATITISVLDAPSRSRSNGSRHLGVATSYLDSLSV 901GDHLQVTVRKNPSSGFRLPSEPETTPMICIAAGSGIAPFRAFLQERAVMMEQDKDRKLAP 961ALLFFGCRAPGIDDLYREQLEEWQARGVVDARWAFSRQSDDTKGCRHVDDRILADREDVV 1021KLWRDGARVYVCGSGALAQSVRSAMVTVLRDEMETTGDGSDNGKAEKWFDEQRNVRYVMD 1081VFD SEQIDNO:34 CYP505A3 A.oryzaeATCC42149 UniprotAccessionNo.Q2U4F1 >gi|121928062|sp|Q2U4F1|Q2U4F1_ASPORCytochromeP450 1MRQNDNEKQICPIPGPQGLPFLGNILDIDLDNGTMSTLKIAKTYYPIFKFTFAGETSIVI 61NSVALLSELCDETRFHKHVSFGLELLRSGTHDGLFTAYDHEKNWELAHRLLVPAFGPLRI 121REMFPQMHDIAQQLCLKWQRYGPRRPLNLVDDFTRTTLDTIALCAMGYRFNSFYSEGDFH 181PFIKSMVRFLKEAETQATLPSFISNLRVRAKRRTQLDIDLMRTVCREIVTERRQTNLDHK 241NDLLDTMLTSRDSLSGDALSDESIIDNILTFLVAGHETTSGLLSFAVYYLLTTPDAMAKA 301AHEVDDVVGDQELTIEHLSMLKYLNAILRETLRLMPTAPGFSVTPYKPEIIGGKYEVKPG 361DSLDVFLAAVHRDPAVYGSDADEFRPERMSDEHFQKLPANSWKPFGNGKRSCIGRAFAWQ 421EALMILALILQSFSLNLVDRGYTLKLKESLTIKPDNLWAYATPRPGRNVLHTRLALQTNS 481THPEGLMSLKHETVESQPATILYGSNSGTCEALAHRLAIEMSSKGRFVCKVQPMDAIEHR 541RLPRGQPVIIITGSYDGRPPENARHFVKWLQSLKGNDLEGIQYAVFGCGLPGHHDWSTTF 601YKIPTLIDTIMAEHGGARLAPRGSADTAEDDPFAELESWSERSVWPGLEAAFDLVRHNSS 661DGTGKSTRITIRSPYTLRAAHETAVVHQVRVLTSAETTKKVHVELALPDTINYRPGDHLA 721ILPLNSRQSVQRVLSLFQIGSDTILYMTSSSATSLPTDTPISAHDLLSGYVELNQVATPT 781SLRSLAAKATDEKTAEYLEALATDRYTTEVRGNHLSLLDILESYSVPSIEIQHYIQMLPL 841LRPRQYTISSSPRLNRGQASLTVSVMERADVGGPRNCAGVASNYLASCTPGSILRVSLRQ 901ANPDFRLPDESCSHPIIMVAAGSGIAPFRAFVQERSVRQKEGIILPPAFLFFGCRRADLD 961DLYREELDAFEEQGVVTLFRAFSRAQSESHGCKYVQDLLWMERVRVKTLWGQDAKVFVCG 1021SVRMNEGVKAIISKIVSPTPTEELARRYIAETFI SEQIDNO:35 CYPX A.oryzaeATCC42149 UniprotAccessionNo.Q2UNA2 >gi|121938553|sp|Q2UNA2|Q2UNA2_ASPORCytochromeP450 1MSTPKAEPVPIPGPRGVPLMGNILDIESEIPLRSLEMMADTYGPIYRLTTFGFSRCMISS 61HELAAEVFDEERFTKKIMAGLSELRHGIHDGLFTAHMGEENWEIAHRVLMPAFGPLNIQN 121MFDEMHDIATQLVMKWARQGPKQKIMVTDDFTRLTLDTIALCAMGTRFNSFYSEEMHPFV 181DAMVGMLKTAGDRSRRPGLVNNLPTTENNKYWEDIDYLRNLCKELVDTRKKNPTDKKDLL 241NALINGRDPKTGKGMSYDSIIDNMITFLIAGHETTSGSLSFAFYNMLKNPQAYQKAQEEV 301DRVIGRRRITVEDLQKLPYITAVMRETLRLTPTAPAIAVGPHPTKNHEDPVTLGNGKYVL 361GKDEPCALLLGKIQRDPKVYGPDAEEFKPERMLDEHFNKLPKHAWKPFGNGMRACIGRPF 421AWQEALLVIAMLLQNFNFQMDDPSYNIQLKQTLTIKPNHFYMRAALREGLDAVHLGSALS 481ASSSEHADHAAGHGKAGAAKKGADLKPMHVYYGSNTGTCEAFARRLADDATSYGYSAEVE 541SLDSAKDSIPKNGPVVFITASYEGQPPDNAAHFFEWLSALKGDKPLDGVNYAVFGCGHHD 601WQTTFYRIPKEVNRLVGENGANRLCEIGLADTANADIVTDFDTWGETSFWPAVAAKFGSN 661TQGSQKSSTFRVEVSSGHRATTLGLQLQEGLVVENTLLTQAGVPAKRTIRFKLPTDTQYK 721CGDYLAILPVNPSTVVRKVMSRFDLPWDAVLRIEKASPSSSKHISIPMDTQVSAYDLFAT 781YVELSQPASKRDLAVLADAAAVDPETQAELQAIASDPARFAEISQKRISVLDLLLQYPSI 841NLAIGDFVAMLPPMRVRQYSISSSPLVDPTECSITFSVLKAPSLAALTKEDEYLGVASTY 901LSELRSGERVQLSVRPSHTGFKPPTELSTPMIMACAGSGLAPFRGFVMDRAEKIRGRRSS 961GSMPEQPAKAILYAGCRTQGKDDIHADELAEWEKIGAVEVRRAYSRPSDGSKGTHVQDLM 1021MEDKKELIDLFESGARIYVCGTPGVGNAVRDSIKSMFLERREEIRRIAKEKGEPVSDDDE 1081ETAFEKFLDDMKTKERYTTDIFA SEQIDNO:36 CYP505A1 F.oxysporum UniprotAccessionNo.Q9Y8G7 >gi|22653677|sp|Q9Y8G7.1|C505_FUSOXRecName:Full= BifunctionalP-450:NADPH- P450reductase;AltName:Full= CytochromeP450foxy;AltName:Full= Fattyacid omega-hydroxylase;Includes:RecName:Full= CytochromeP450505;Includes: RecName:Full= NADPH--cytochromeP450reductase 1maesvpipeppgyplignlgeftsnplsdlnrladtygpifrlrlgakapifvssnslin 61evcdekrfkktlksvlsqvregvhdglftafedepnwgkahrilvpafgplsirgmfpem 121hdiatqlcmkfarhgprtpidtsdnftrlaldtlalcamdfrfysyykeelhpfieamgd 181fltesgnrnrrppfapnflyraanekfygdialmksvadevvaarkaspsdrkdllaaml 241ngvdpqtgeklsdenitnqlitfliaghettsgtlsfamyqllknpeayskvqkevdevv 301grgpvlvehltklpyisavlretlrinspitafgleaiddtflggkylvkkgeivtalls 361rghvdpvvygndadkfipermlddefarinkeypncwkpfgngkracigrpfawqeslla 421mvvlfqnfnftmtdpnyaleikqtltikpdhfyinatlrhgmtptelehvlagngatsss 481thnikaaanldakagsgkpmaifygsnsgtcealanrlasdapshgfsattvgpldqakq 541nlpedrpvvivtasyegqppsnaahfikwmedldgndmekvsyavfacghhdwvetfhri 601pklvdstlekrggtrlvpmgsadaatsdmfsdfeawedivlwpglkekykisdeesggqk 661gllvevstprktslrqdveealvvaektltksgpakkhieiqlpsamtykagdylailpl 721npkstvarvfrrfslawdsflkiqsegptt1ptnvaisafdvfsayvelsqpatkrnila 781laeatedkdtiqelerlagdayqaeispkrvsvldllekfpavalpissylamlppmrvr 841qysissspfadpskltltyslldapslsgqgrhvgvatnflshltagdklhvsvrassea 901fhlpsdaektpiicvaagtglaplrgfiqeraamlaagrtlapallffgcrnpeiddlya 961eeferwekmgavdvrraysratdksegckyvqdrvyhdradvfkvwdqgakvficgsrei 1021gkavedvcvrlaiekaqqngrdvteemarawfersrnerfatdvfd SEQIDNO:37 CYPX G.moniliformis GenBankAccessionNo.AAG27132 >gi|11035011|gb|AAG27132.1|Fum6p[Fusariumverticillioides] 1MSATALFTRRSVSTSNPELRPIPGPKPLPLLGNLFDFDFDNLTKSLGELGKIHGPIYSIT 61FGASTEIMVTSREIAQELCDETRFCKLPGGALDVMKAVVGDGLFTAETSNPKWAIAHRII 121TPLFGAMRIRGMFDDMKDICEQMCLRWARFGPDEPLNVCDNMTKLTLDTIALCTIDYRFN 181SFYRENGAAHPFAEAVVDVMTESFDQSNLPDFVNNYVRFRAMAKFKRQAAELRRQTEELI 241AARRQNPVDRDDLLNAMLSAKDPKTGEGLSPESIVDNLLTFLIAGHETTSSLLSFCFYYL 301LENPHVLRRVQQEVDTVVGSDTITVDHLSSMPYLEAVLRETLRLRDPGPGFYVKPLKDEV 361VAGKYAVNKDQPLFIVFDSVHRDQSTYGADADEFRPERMLKDGFDKLPPCAWKPFGNGVR 421ACVGRPFAMQQAILAVAMVLHKFDLVKDESYTLKYHVTMTVRPVGFTMKVRLRQGQRATD 481LAMGLHRGHSQEASAAASPSRASLKRLSSDVNGDDTDHKSQIAVLYASNSGSCEALAYRL 541AAEATERGFGIRAVDVVNNAIDRIPVGSPVILITASYNGEPADDAQEFVPWLKSLESGRL 601NGVKFAVFGNGHRDWANTLFAVPRLIDSELARCGAERVSLMGVSDTCDSSDPFSDFERWI 661DEKLFPELETPHGPGGVKNGDRAVPRQELQVSLGQPPRITMRKGYVRAIVTEARSLSSPG 721VPEKRHLELLLPKDFNYKAGDHVYILPRNSPRDVVRALSYFGLGEDTLITIRNTARKLSL 781GLPLDTPITATDLLGAYVELGRTASLKNLWTLVDAAGHGSRAALLSLTEPERFRAEVQDR 841HVSILDLLERFPDIDLSLSCFLPMLAQIRPRAYSFSSAPDWKPGHATLTYTVVDFATPAT 901QGINGSSKSKAVGDGTAVVQRQGLASSYLSSLGPGTSLYVSLHRASPYFCLQKSTSLPVI 961MVGAGTGLAPFRAFLQERRMAAEGAKQRFGPALLFFGCRGPRLDSLYSVELEAYETIGLV 1021QVRRAYSRDPSAQDAQGCKYVTDRLGKCRDEVARLWMDGAQVLVCGGKKMANDVLEVLGP 1081MLLEIDQKRGETTAKTVVEWRARLDKSRYVEEVYV SEQIDNO:38 CYP505A7 G.zeaePH1 GenBankAccessionNo.EAA67736 >gi|42544893|gb|EAA67736.1|C505_FUSOXBifunctionalP-450:NADPH-P450 reductase(Fattyacidomega-hydroxylase)(P450foxy)[GibberellazeaePH-1] 1MAESVPIPEPPGYPLIGNLGEFKTNPLNDLNRLADTYGPIFRLHLGSKTPTFVSSNAFIN 61EVCDEKRFKKTLKSVLSVVREGVHDGLFTAFEDEPNWGKAHRILIPAFGPLSIRNMFPEM 121HEIANQLCMKLARHGPHTPVDASDNFTRLALDTLALCAMDFRFNSYYKEELHPFIEAMGD 181FLLESGNRNRRPAFAPNFLYRAANDKFYADIALMKSVADEVVATRKQNPTDRKDLLAAML 241EGVDPQTGEKLSDDNITNQLITFLIAGHETTSGTLSFAMYHLLKNPEAYNKLQKEIDEVI 301GRDPVTVEHLTKLPYLSAVLRETLRISSPITGFGVEAIEDTFLGGKYLIKKGETVLSVLS 361RGHVDPVVYGPDAEKFVPERMLDDEFARLNKEFPNCWKPFGNGKRACIGRPFAWQESLLA 421MALLFQNFNFTQTDPNYELQIKQNLTIKPDNEFFNCTLRHGMTPTDLEGQLAGKGATTSI 481ASHIKAPAASKGAKASNGKPMAIYYGSNSGTCEALANRLASDAAGHGFSASVIGTLDQAK 541QNLPEDRPVVIVTASYEGQPPSNAAHFIKWMEDLAGNEMEKVSYAVFGCGHHDWVDTFLR 601IPKLVDTTLEQRGGTRLVPMGSADAATSDMFSDFEAWEDTVLWPSLKEKYNVTDDEASGQ 661RGLLVEVTTPRKTTLRQDVEEALVVSEKTLTKTGPAKKHIEIQLPSGMTYKAGDYLAILP 721LNPRKTVSRVFRRFSLAWDSFLKIQSDGPTTLPINIAISAFDVFSAYVELSQPATKRNIL 781ALSEATEDKATIQELEKLAGDAYQEDVSAKKVSVLDLLEKYPAVALPISSYLAMLPPMRV 841RQYSISSSPFADPSKLTLTYSLLDAPSLSGQGRHVGVATNFLSQLIAGDKLHISVRASSA 901AFHLPSDPETTPIICVAAGTGLAPFRGFIQERAAMLAAGRKLAPALLFFGCRDPENDDLY 961AEELARWEQMGAVDVRRAYSRATDKSEGCKYVQDRIYHDRADVFKVWDQGAKVFICGSRE 1021IGKAVEDICVRLAMERSEATQEGKGATEEKAREWFERSRNERFATDVFD SEQIDNO:39 CYP505C2 G.zeaePHla GenBankAccessionNo.EAA77183 >gi|42554340|gb|EAA77183.1|hypotheticalproteinFG07596.1[Gibberellazeae PH-1] 1MAIKDGGKKSGQIPGPKGLPVLGNLFDLDLSDSLTSLINIGQKYAPIFSLELGGHREVMI 61CSRDLLDELCDETRFHKIVTGGVDKLRPLAGDGLFTAQHGNHDWGIAHRILMPLFGPLKI 121REMFDDMQDVSEQLCLKWARLGPSATIDVANDFTRLTLDTIALCTMGYRFNSFYSNDKMH 181PFVDSMVAALIDADKQSMFPDFIGACRVKALSAFRKHAAIMKGTCNELIQERRKNPIEGT 241DLLTAMMEGKDPKTGEGMSDDLIVQNLITFLIAGHETTSGLLSFAFYYLLENPHTLEKAR 301AEVDEVVGDQALNVDHLTKMPYVNMILRETLRLMPTAPGFFVTPHKDEIIGGKYAVPANE 361SLFCFLHLIHRDPKVWGADAEEFRPERMADEFFEALPKNAWKPFGNGMRGCIGREFAWQE 421AKLITVMILQNFELSKADPSYKLKIKQSLTIKPDGFNMHAKLRNDRKVSGLFKAPSLSSQ 481QPSLSSRQSINAINAKDLKPISIFYGSNTGTCEALAQKLSADCVASGFMPSKPLPLDMAT 541KNLSKDGPNILLAASYDGRPSDNAEEFTKWAESLKPGELEGVQFAVFGCGHKDWVSTYFK 601IPKILDKCLADAGAERLVEIGLTDASTGRLYSDFDDWENQKLFTELSKRQGVTPTDDSHL 661ELNVTVIQPQNNDMGGNFKRAEVVENTLLTYPGVSRKHSLLLKLPKDMEYTPGDHVLVLP 721KNPPQLVEQAMSCFGVDSDTALTISSKRPTFLPTDTPILISSLLSSLVELSQTVSRTSLK 781RLADFADDDDTKACVERIAGDDYTVEVEEQRMSLLDILRKYPGINMPLSTFLSMLPQMRP 841RTYSFASAPEWKQGHGMLLFSVVEAEEGTVSRPGGLATNYMAQLRQGDSILVEPRPCRPE 901LRTTMMLPEPKVPIIMIAVGAGLAPFLGYLQKRFLQAQSQRTALPPCTLLFGCRGAKMDD 961ICRAQLDEYSRAGVVSVHRAYSRDPDSQCKYVQGLVTKHSETLAKQWAQGAIVMVCSGKK 1021VSDGVMNVLSPILFAEEKRSGMTGADSVDVWRQNVPKERMILEVFG SEQIDNO:40 CYP505A5 M.grisea70-15syn GenBankAccessionNo.XP365223 >gi|145601517|ref|XP_365223.21hypotheticalproteinMGG01925[Magnaporthe oryzae70-15] 1MFFLSSSLAYMAATQSRDWASFGVSLPSTALGRHLQAAMPFLSEENHKSQGTVLIPDAQG 61PIPFLGSVPLVDPELPSQSLQRLARQYGEIYRFVIPGRQSPILVSTHALVNELCDEKRFK 121KKVAAALLGLREAIHDGLFTAHNDEPNWGIAHRILMPAFGPMAIKGMFDEMHDVASQMIL 181KWARHGSTTPIMVSDDFTRLTLDTIALCSMGYRFNSFYHDSMHEFIEAMTCWMKESGNKT 241RRLLPDVFYRTTDKKWHDDAEILRRTADEVLKARKENPSGRKDLLTAMIEGVDPKTGGKL 301SDSSIIDNLITFLIAGHETTSGMLSFAFYLLLKNPTAYRKAQQEIDDLCGREPITVEHLS 361KMPYITAVLRETLRLYSTIPAFVVEAIEDTVVGGKYAIPKNHPIFLMIAESHRDPKVYGD 421DAQEFEPERMLDGQFERRNREFPNSWKPFGNGMRGCIGRAFAWQEALLITAMLLQNFNFV 481MHDPAYQLSIKENLTLKPDNFYMRAILRHGMSPTELERSISGVAPTGNKTPPRNATRTSS 541PDPEDGGIPMSIYYGSNSGTCESLAHKLAVDASAQGFKAETVDVLDAANQKLPAGNRGPV 601VLITASYEGLPPDNAKHFVEWLENLKGGDELVDTSYAVFGCGHQDWTKTFHRIPKLVDEK 661LAEHGAVRLAPLGLSNAAHGDMFVDFETWEFETLWPALADRYKTGAGRQDAAATDLTAAL 721SQLSVEVSHPRAADLRQDVGEAVVVAARDLTAPGAPPKRHMEIRLPKTGGRVHYSAGDYL 781AVLPVNPKSTVERAMRRFGLAWDAHVTIRSGGRTTLPTGAPVSAREVLSSYVELTQPATK 841RGIAVLAGAVTGGPAAEQEQAKAALLDLAGDSYALEVSAKRVGVLDLLERFPACAVPFGT 901FLALLPPMRVRQYSISSSPLWNDEHATLTYSVLSAPSLADPARTHVGVASSYLAGLGEGD 961HLHVALRPSHVAFRLPSPETPVVCVCAGSGMAPFRAFAQERAALVGAGRKVAPLLLFFGC 1021REPGVDDLYREELEGWEAKGVLSVRRAYSRRTEQSEGCRYVQDRLLKNRAEVKSLWSQDA 1081KVFVCGSREVAEGVKEAMFKVVAGKEGSSEEVQAWYEEVRNVRYASDIFD SEQIDNO:41 CYP505A2 N.crassa0R74A GenBankAccessionNo.XP961848 >gi|85104987|ref|XP_961848.1|bifunctionalP-450:NADPH-P450reductase [NeurosporacrassaOR74A] 1MSSDETPQTIPIPGPPGLPLVGNSFDIDTEFPLGSMLNFADQYGEIFRLNFPGRNTVFVT 61SQALVHELCDEKRFQKTVNSALHEIRHGIHDGLFTARNDEPNWGIAHRILMPAFGPMAIQ 121NMFPEMHEIASQLALKWARHGPNQSIKVTDDFTRLTLDTIALCSMDYRFNSYYHDDMHPF 181IDAMASFLVESGNRSRRPALPAFMYSKVDRKFYDDIRVLRETAEGVLKSRKEHPSERKDL 241LTAMLDGVDPKTGGKLSDDSIIDNLITFLIAGHETTSGLLSFAFVQLLKNPETYRKAQKE 301VDDVCGKGPIKLEHMNKLHYIAAVLRETLRLCPTIPVIGVESKEDTVIGGKYEVSKGQPF 361ALLFAKSHVDPAVYGDTANDFDPERMLDENFERLNKEFPDCWKPFGNGMRACIGRPFAWQ 421EALLVMAVCLQNFNFMPEDPNYTLQYKQTLTTKPKGFYMRAMLRDGMSALDLERRLKGEL 481VAPKPTAQGPVSGQPKKSGEGKPISIYYGSNTGTCETFAQRLASDAEAHGFTATIIDSLD 541AANQNLPKDRPVVFITASYEGQPPDNAALFVGWLESLTGNELEGVQYAVFGCGHHDWAQT 601FHRIPKLVDNTVSERGGDRICSLGLADAGKGEMFTEFEQWEDEVFWPAMEEKYEVSRKED 661DNEALLQSGLTVNFSKPRSSTLRQDVQEAVVVDAKTITAPGAPPKRHIEVQLSSDSGAYR 721SGDYLAVLPINPKETVNRVMRRFQLAWDTNITIEASRQTTILPTGVPMPVHDVLGAYVEL 781SQPATKKNILALAEAADNAETKATLRQLAGPEYTEKITSRRVSILDLLEQFPSIPLPFSS 841FLSLLPPMRVRQYSISSSPLWNPSHVTLTYSLLESPSLSNPDKKHVGVATSYLASLEAGD 901KLNVSIRPSHKAFHLPVDADKTPLIMIAAGSGLAPFRGFVQERAAQIAAGRSLAPAMLFY 961GCRHPEQDDLYRDEFDKWESIGAVSVRRAFSRCPESQETKGCKYVGDRLWEDREEVTGLW 1021DRGAKVYVCGSREVGESVKKVVVRIALERQKMIVEAREKGELDSLPEGIVEGLKLKGLTV 1081EDVEVSEERALKWFEGIRNERYATDVFD SEQIDNO:42 CYP97C Oryzasativa GenBankAccessionNo.ABB47954 >gi|78708979|gb|ABB47954.1|CytochromeP450familyprotein,expressed [OryzasativaJaponicaGroup] 1MAAAAAAAVPCVPFLCPPPPPLVSPRLRRGHVRLRLRPPRSSGGGGGGGAGGDEPPITTS 61WVSPDWLTALSRSVATRLGGGDDSGIPVASAKLDDVRDLLGGALFLPLFKWFREEGPVYR 121LAAGPRDLVVVSDPAVARHVLRGYGSRYEKGLVAEVSEFLFGSGFAIAEGALWTVRRRSV 181VPSLHKRFLSVMVDRVFCKCAERLVEKLETSALSGKPVNMEARFSQMTLDVIGLSLFNYN 241FDSLTSDSPVIDAVYTALKEAELRSTDLLPYWKIDLLCKIVPRQIKAEKAVNIIRNTVED 301LITKCKKIVDAENEQIEGEEYVNEADPSILRFLLASREEVTSVQLRDDLLSMLVAGHETT 361GSVLTWTIYLLSKDPAALRRAQAEVDRVLQGRLPRYEDLKELKYLMRCINESMRLYPHPP 421VLIRRAIVDDVLPGNYKIKAGQDIMISVYNIHRSPEVWDRADDFIPERFDLEGPVPNETN 481TEYRFIPFSGGPRKCVGDQFALLEAIVALAVVLQKMDIELVPDQKINMTTGATIHTTNGL 541YMNVSLRKVDREPDFALSGSR SEQIDNO:43 ChimerichemeenzymeC2G9 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEG ALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLIQKWARLNPNEAVDVPGDMTRL TLDTIGLCGFNYRFNSYYRETPHPFINSMVRALDEAMHQMQRLDVQDKLMVRTKRQFRYDIQTMFSLVDRMIAER KANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARV LVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAE DERPERFEDPSSIPHHAYKPFGNGQRACIGMQFALHEATLVLGMILKYFTLIDHENYELDIKQTLTLKPGDFHIS VQSRHQEAIHADVQAAE SEQIDNO:44 ChimerichemeenzymeX7 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEG ALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRL TLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAER RANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRV LTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAE DERPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKIT VKPRKTAAINVQRKEQA SEQIDNO:45 ChimerichemeenzymeX7-12 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDEERFDKSIEGA LEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLVQKWERLNADEHIEVPEDMTRLT LDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERR ANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVL TDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAED FRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITV KPRKTAAINVQRKEQA SEQIDNO:46 ChimerichemeenzymeC2E6 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQA LKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLT LDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERK ANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVL TDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEE FRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLTLKPEGFVVKA KSKKIPLGGIPSPST SEQIDNO:47 ChimerichemeenzymeX7-9 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFRFDFPGVSSVFVSGHNLVAEVCDEERFDKSIEG ALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTFSQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRL TLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAER RANGDQDEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRV LTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAE DFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKIT VKPRKTAAINVQRKEQA SEQIDNO:48 ChimerichemeenzymeC2B12 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFRFDFPGVSSVFVSGHNLVAEVCDEERFDKSIEG ALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRL TLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAER KANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRV LTDAAPTYKQVLELTYIRMILNESLRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAE DERPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKIT VKPRKTAAINVQRKEQA SEQIDNO:49 ChimerichemeenzymeT5P234 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEG ALEKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRL TLDTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAER KANPDENIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRV LTDDTPEYKQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAE DERPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKIT VKPRKTAAINVQRKEQA SEQIDNO:50 WT-AxA(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRAAIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:51 WT-AxD(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRADIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:52 WT-AxH(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGENYRENSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRAHIGQQFALHEATLVLGMMLKHEDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:53 WT-AxK(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRAKIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:54 WT-AxM(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRAMIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:55 WT-AxN(heme) TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDKIIADRKA SGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVD PVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFR PERFENPSAIPQHAFKPFGNGQRANIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKS KKIPLGGIPSPST SEQIDNO:56 BM3-CIS-T4385-AxA TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDIIADRKAR GEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYFLVKNPHVLQKVAEEAARVLVDP VPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRP ERFENPSAIPQHAFKPFGNGQRAAIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKSK KIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPRE GAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRG EADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPG SARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQ YVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRP RYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMV GPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQ HVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:57 BM3-CIS-T4385-AxD TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDIIADRKAR GEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYFLVKNPHVLQKVAEEAARVLVDP VPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRP ERFENPSAIPQHAFKPFGNGQRADIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKSK KIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPRE GAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRG EADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPG SARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQ YVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRP RYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMV GPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQ HVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:58 BM3-CIS-T438S-AxM TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDIIADRKAR GEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYFLVKNPHVLQKVAEEAARVLVDP VPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRP ERFENPSAIPQHAFKPFGNGQRAMIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKSK KIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPRE GAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRG EADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPG SARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQ YVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRP RYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMV GPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQ HVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:59 BM3-CIS-T4385-AxY TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDIIADRKAR GEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYFLVKNPHVLQKVAEEAARVLVDP VPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRP ERFENPSAIPQHAFKPFGNGQRAYIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKSK KIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPRE GAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRG EADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPG SARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQ YVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRP RYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMV GPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQ HVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:60 BM3-CIS-T4385-AxT TIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQAL KFARDFAGDGLVTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVSEDMTRLTL DTIGLCGFNYRFNSFYRDQPHPFIISMVRALDEVMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDIIADRKAR GEQSDDLLTQMLNGKDPETGEPLDDGNIRYQIITFLIAGHEATSGLLSFALYFLVKNPHVLQKVAEEAARVLVDP VPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEKGDEVMVLIPQLHRDKTVWGDDVEEFRP ERFENPSAIPQHAFKPFGNGQRATIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLSLKPKGFVVKAKSK KIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPRE GAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRG EADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPG SARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQ YVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRP RYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMV GPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQ HVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQIDNO:61 M.infernorumHemoglobinI 1MIDQKEKELIKESWKRIEPNKNEIGLLFYANLFKEEPTVSVLFQNPISSQ 51SRKLMQVLGILVQGIDNLEGLIPTLQDLGRRHKQYGVVDSHYPLVGDCLL 101KSIQEYLGQGFTEEAKAAWTKVYGIAAQVMTAE SEQIDNO:62 Bacillussubtilistruncatedhemoglobin 1MGQSFNAPYEAIGEELLSQLVDTFYERVASHPLLKPIFPSDLTETARKQK 51QFLTQYLGGPPLYTEEHGHPMLRARHLPFPITNERADAWLSCMKDAMDHV 101GLEGEIREFLFGRLELTARHMVNQTEAEDRSS