Patent Publication Number: US-2016222423-A1

Title: Enzyme-catalyzed enantioselective aziridination of olefins

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS 
     The present application claims priority to U.S. Provisional Patent Application No. 62/108,300, filed Jan. 27, 2015, and U.S. Provisional Patent Application No. 62/120,126, filed Feb. 24, 2015, the contents of which are hereby incorporated by reference in their entirety for all purposes. 
    
    
     STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT 
     This invention was made with government support under Grant No. N00014-11-1-0205 awarded by the Office of Naval Research. The government has certain rights in the invention. 
    
    
     REFERENCE TO A SEQUENCE LISTING 
     The Sequence Listing written in file 086544-019120US-0966192_SequenceListing.txt, created on Apr. 11, 2016, 418,993 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes. 
     BACKGROUND OF THE INVENTION 
     Aziridines are 3 membered cyclic compounds comprising 2 carbons and a nitrogen that are often used as building blocks in various synthetic strategies. Traditional synthesis of aziridines can be achieved through various known methods; however, many of these method use caustic chemicals, harsh reaction conditions, and/or are unable to produce stereo-selective chiral aziridines. 
     Enzymes offer appealing alternatives to traditional chemical catalysts due to their ability to function in aqueous media at ambient temperature and pressure, as well as their ability to orient substrate binding for defined regio- and stereo-chemical outcomes. Indeed, the use of enzymes in synthetic chemistry to achieve otherwise difficult or low yielding chemical conversions is continuing to increase. 
     Although chemically attractive, enzymes are also known for their high substrate specificity and their catalytic fidelity. While this selectivity can be advantageous in some cases, it is also a significant synthetic limitation because each enzyme typically catalyzes only a specific chemical reaction. Despite these limitations, previous studies have shown that the native activity of enzymes can be modified or altered to catalyze non-natural or non-naturally occurring chemical conversions. Development of an enzyme capable of catalyzing an aziridination reaction could avoid using caustic chemicals, harsh reaction conditions, and could reliably produce stereo-selective chiral aziridines. 
     As such, there is a need in the art for novel reagents and catalytic schemes that are capable of creating an aziridine functional group with high yield, regioselectivity, and stereoselectivity, but without the toxicity and harsh reaction conditions associated with current approaches. The present invention addresses these and other needs. 
     BRIEF SUMMARY OF THE INVENTION 
     In a first aspect, the invention provides a reaction mixture for producing an aziridination product. The reaction mixture includes an olefinic substrate, a nitrene precursor, and a heme enzyme. 
     In some embodiments, the olefinic substrate is represented by a structure of Formula I: 
     
       
         
         
             
             
         
       
     
     wherein:
         R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-8 alkyl, C 1-8 heteroalkyl, aryl, heteroaryl, C 1-12 cycloalkyl, C 3-10 heterocyclyl, —Y 1 -aryl, —Y 1 -heteroaryl, —Y 1 —C 1-12 cycloalkyl and —Y 1 —C 3-10 heterocyclyl;   Y 1  is C 1-8 alkylene;   each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo, cyano, and halogen;   wherein each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 8 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S.       

     In some instances, R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, aryl, heteroaryl, C 1-12 cycloalkyl, and C 3-10 heterocyclyl, and each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy, and halogen. 
     In some embodiments, the nitrene precursor has a structure according to Formula IIa or IIb: 
     
       
         
         
             
             
         
       
     
     wherein:
         R 3  is selected from the group consisting of C 1-18  alkyl, C 1-8 heteroalkyl, C 3-12 cycloalkyl, aryl, heteroaryl, C 3-10 heterocyclyl, —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ;   X 1  is independently selected from the group consisting of H and sodium, and   X 2  is independently selected from the group consisting of halogen, —SO 2 R a , —CO 2 R b , —PO 3 R b R c , optionally X 1  and X 2  can be taken together to form iodinane;   R a  is independently selected from the group consisting of C 1-8 alkyl, hydroxy, C 1-8 alkoxy, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents;   each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy; and   wherein each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S.       

     In some embodiments, the nitrene precursor has a structure selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     In some instances, the nitrene precursor is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the aziridination product is produced in vitro. 
     In some embodiments, the reaction mixture further comprises a reducing agent. In some instances, the reducing agent is NADPH. 
     In some embodiments, the heme the heme enzyme is localized within a whole cell and the aziridination product is produced in vivo. In some instances, the whole cell is a bacterial cell or a yeast cell. 
     In some embodiments, the aziridination product is produced under anaerobic conditions. 
     In some embodiments, the heme enzyme is a variant thereof comprising a mutation at the axial position of the heme coordination site. In some instances, the heme enzyme comprises a serine mutation at the axial position of the heme coordination site. 
     In some embodiments, the heme enzyme is a cytochrome P450 enzyme or a variant thereof. In some instances, the cytochrome P450 enzyme is a P450 BM3 enzyme or a variant thereof. 
     In some embodiments, the P450 BM3 enzyme comprises an axial ligand mutation C400S and one or more mutations selected from the group consisting of V78, F87, P142, T175, A184, S226, H236, E252, I263, T268, A290, A328, L353, I366, L437, T438, and E442 relative to the amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 50). In some instances, the P450 BM3 enzyme comprises an axial ligand mutation C400S and mutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, A328V, L353V, I366V, L437V, T438S, and E442K relative to the amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 51). 
     In some embodiments, the P450 BM3 enzyme comprises an axial ligand mutation C400S and one or more mutations selected from the group consisting of L75, V78, F87, P142, T175, L181, A184, S226, H236, E252, I263, T268, A290, L353, I366, and E442 relative to the amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 52). In some instances, the P450 BM3 enzyme comprises an axial ligand mutation C400S and mutations L75A, V78A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, and E442K relative to the amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 53). 
     In some embodiments, the aziridination product is an aziridine compound according to Formula III: 
     
       
         
         
             
             
         
       
     
     wherein
         R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, C 1-8 heteroalkyl, aryl, heteroaryl, C 1-12 cycloalkyl, C 3-10 heterocyclyl, —Y 1 -aryl, —Y 1 -heteroaryl, —Y 1 —C 1-12 cycloalkyl and —Y 1 —C 3-10 heterocyclyl;   Y 1  is C 1-8 alkylene;   each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo, cyano, and halogen;   R 3  is selected from the group consisting of C 1-18  alkyl, C 1-8 heteroalkyl, C 3-12 cycloalkyl, aryl, heteroaryl, C 3-10 heterocyclyl, —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ;   R a  is independently selected from the group consisting of C 1-8 alkyl, hydroxy, C 1-8 alkoxy, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents;   each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy; and   wherein each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S.       

     In some instances, R 1a  and R 1b  are independently selected from the group consisting of H, C 1-8 alkyl, aryl, heteroaryl, C 1-12 cycloalkyl, and C 3-10 heterocyclyl;
         R 2  is selected from the group consisting of H and C 1-8  alkyl;   each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 3 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy, and halogen; and   R 3  is selected from the group consisting of —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ,   R a  is independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   R b  and R c  are independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl;   wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-2 R d  substituents; and   each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy.       

     In some embodiments, the aziridination product is an amido-alcohol compound according for Formula IIIa: 
     
       
         
         
             
             
         
       
     
     wherein R 1a , R 1b , R 3 , and R 3  can be as defined above in Formula III. 
     In some embodiments, the reaction produces a plurality of aziridination products. In some instances, the plurality of aziridination products has a % ee S  of from about −99% to about 99%. In some instances, the plurality of aziridination products has a % ee S  of from about −81% to about 81%. In some instances, the plurality of aziridination products has a Z:E ratio of from about 1:99 to about 99:1. 
     In some aspects, the present invention provides a cytochrome P450 BM3 enzyme variant or fragment thereof that can a aziridinate an olefinic substrate comprising an axial ligand mutation C400S, mutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, T438S, and E442K, and at least one or more mutations at positions A328 and/or L437 relative to the amino acid sequence set forth in SEQ ID NO:1 (SEQ ID NO: 54). In some instances, the cytochrome P450 BM3 enzyme variant comprises an axial ligand mutation C400S and mutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, A328V, L353V, I366V, L437V, T438S, and E442K relative to the amino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 51). 
     In some embodiments, the cytochrome P450 BM3 enzyme variant produces a plurality of aziridination products with a % ee S  of at least about 75%. In some instances, the enzyme variant has a higher total turnover number (TTN) compared to the wild-type sequence. In some instances, the enzyme variant has a TTN greater than about 100. 
     In another aspect, the present invention provides a cytochrome P450 BM3 enzyme variant or fragment thereof that can aziridinate an olefinic substrate comprising an axial ligand mutation C400S, mutations L75A, V87A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, and E442K, and a mutation at position I263 relative to the amino acid sequence set forth in SEQ ID NO: 1. In some instances, the enzyme variant comprises an axial ligand mutation C400S and mutations L75A, V78A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, and E442K relative to the amino acid sequence set forth in SEQ ID NO: 1 (SEQ ID NO: 53). 
     In some embodiments, the cytochrome P450 BM3 enzyme variant produces a plurality of aziridination products with a % ee R  of at least about 75%. In some instances, the enzyme variant has a higher total turnover number (TTN) compared to the wild-type sequence. In some instances, the enzyme variant has a TTN greater than about 100. 
     In certain aspects, the present invention provides a method for producing an aziridination product, the method comprising:
         (a) providing an olefinic substrate, a nitrene precursor, and a heme enzyme; and   (b) admixing the components of step (a) in a reaction for a time sufficient to produce an aziridination product.       

     In some embodiments, the method produces a plurality of aziridination products. In some instances, the plurality of aziridination products has a % ee S  of from about −90% to about 90%. In certain instances, the plurality of aziridination products has a % ee S  of from about −81% to about 81%. In some instances, the plurality of aziridination products has a Z:E ratio of from 1:99 to 99:1. In some instances, the aziridination reaction is at least 30% to at least 90% diastereoselective. 
     In some embodiments, the aziridination product is a compound according to Formula III: 
     
       
         
         
             
             
         
       
     
     wherein R 1a , R 1b , R 3 , and R 3  can be as defined above in Formula III. 
     In certain other embodiments, the aziridination product is a compound according to Formula IIIa: 
     
       
         
         
             
             
         
       
     
     wherein R 1a , R 1b , R 3 , and R 3  can be as defined above in Formula III. 
     Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a heme enzyme intermolecular nitrogen-atom transfer in accordance with an embodiment of the invention. 
         FIGS. 2A-B  show HPLC 220 nm chromatograms of controls:  FIG. 2A —Co-injection of 4-methoxystyrene (Sigma Aldrich) and an N-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine synthetic standard, confirmed by NMR;  FIG. 2B —Injection of the N-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine synthetic standard alone. 
         FIGS. 3A-D  show HPLC 220 nm chromatograms of P411-enzymatic reactions with 4-methoxystyrene and tosyl azide as substrates analyzed at different time points. Putative N-(p-Tolyl sulfonyl)-2-(p-methoxyphenyl)aziridine and amido-alcohol derivative ((N-(2-hydroxy-2-(4-methoxyphenyl)ethyl)-4-methylbenzenesulfonamide, 2) are marked with arrows. 
         FIGS. 4A-D  show HPLC 220 nm chromatograms of synthetic standard S1, synthesized as previously reported, in reaction conditions without P411 catalyst at several time points. Putative aziridine and amido-alcohol are marked with arrows, as in  FIGS. 3A-D . 
         FIG. 5  shows a comparison of P-I263F productivity in vitro (purified protein) and in whole cells. 
         FIG. 6  shows initial rates of aziridination and azide reduction for engineered enzymes. Total turnover (TTN) values were determined using the same method as described for initial rates, with the exception that reactions were allowed to proceed for 4 hours in the anaerobic chamber. 
         FIGS. 7A-C  show data used to determine initial rates for enzymes (A) P-I263F, (B) P-I263F-A328V, and (C) P-I263F-A328V-L437V. Diamonds represent concentrations of tosyl sulfonamide 7 and triangles represent concentrations of aziridine 4 for all plots. 
         FIG. 8  shows activity and selectivity of P-I263F-A328V-L437V with increased substrate loading. Reactions were performed with whole  E. coli  cells expressing P-I263F-A328V-L437V as described in the general methods, except substrate loading was increased to final concentrations of 7.5 mM tosyl azide and 15 mM olefin. 
         FIGS. 9A-B  are an exemplary demonstration of how absolute stereochemistry can be defined for the products herein. The assignment of absolute stereochemistry of enzymatically produced aziridine 6 is assigned using chiral HPLC (Chiracel OJ, 30% isopropanol: 70% n-hexane, 210 nm). Top: Racemic synthetic aziridine 6, t R =16.7 min and 21.0 min; Bottom: P-I263F-A328V-L437V produced aziridine 6, t R =16.8 min. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     I. Introduction 
     The present invention is based, in part, on the surprising discovery that heme enzymes can be used to catalyze the conversion of olefinic bonds to aziridination products in the presence of nitrene precursors.  FIG. 1  illustrates an exemplary reaction where styrene is converted to an aziridination product. In some aspects, cytochrome P450 BM3 enzymes and variants thereof were identified as having an unexpectedly efficient ability to catalyze the formal transfer of nitrene equivalents from nitrene precursors to various olefinic substrates, thereby making aziridination products with high regioselectivity and/or stereoselectivity. In particular embodiments, the present inventors have discovered that variants of P450 BM3 with at least one or more amino acid mutations such as an axial ligand C400S, mutations V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, T438S, and E442K, and at least one or more mutations at positions A328 and/or L437 (SEQ ID NO: 54) can catalyze aziridination reactions efficiently, displaying increased total turnover numbers (TTN) and demonstrating highly regio- and enantioselective product formation compared to wild-type enzymes. 
     Aziridination reactions can be performed by the heme enzymes described herein in vitro or in vivo, where the heme enzyme is localized within a whole cell. In some embodiments, the heme enzyme described herein can catalyze the aziridination reaction in vivo, providing over 500 total turnovers with high stereoselectivity and yield. 
     The disclosure herein highlights the utility of enzymes in catalyzing new types of reactions. The ability to genetically encode catalysts for formal nitrene transfers opens up new biosynthetic pathways to amines and expands the scope of transformations accessible to biocatalysis. 
     II. Definitions 
     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. 
     As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a species” includes a plurality of such species and reference to “the enzyme” includes reference to one or more enzymes and equivalents thereof, and so forth. 
     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.” 
     “About” and “around,” as used herein to modify a numerical value, indicate a defined range around that value. If “X” were the value, “about X” or “around X” would generally indicate 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.” When the quantity “X” only includes whole-integer values (e.g., “X carbons”), “about X” or “around X” indicates from (X−1) to (X+1). In such cases, “about X” or “around X” specifically indicates at least the values X, X−1, and X+1. 
     The term “aziridination (enzyme) catalyst” or “enzyme with aziridination 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 an aziridination product by using nitrene precursors. 
     The terms “engineered heme enzyme” and “heme enzyme variant” include any heme 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 enzymes. 
     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. 
     The term “whole cell catalyst” includes microbial cells expressing at least one engineered heme enzyme, wherein the whole cell catalyst displays aziridination activity. 
     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. 
     The term “heme” or “heme domain” as used herein refers to an amino acid sequence within an enzyme, which is capable of binding an iron-complexing structure such as a porphyrin. Compounds of iron are typically complexed in a porphyrin (tetrapyrrole) ring that may differ in side chain composition. Heme groups can be the prosthetic groups of cytochromes and are found in most oxygen carrier proteins. Exemplary heme domains include that of P450 BM3 as well as truncated or mutated versions of these that retain the capability to bind the iron-complexing structure. A skilled person can identify the heme domain of a specific protein using methods known in the art. 
     The terms “nitrene equivalent” and “nitrene precursor” include molecules that can be decomposed in the presence of metal (or enzyme) catalysts to structures that contain at least one nitrogen with only 5 valence shell electrons and that can be transferred to C═C bonds to form aziridines. Nitrene precursors of the present invention include, but are not limited to, sulfonyl azides, carbonyl azides, aryl azides, azidoformates, phosphoryl azides, azide phosphonates, iminoiodanes, or haloamine derivatives. 
     The terms “nitrene transfer” and “formal nitrene transfer” as used herein include any chemical transformation where nitrene equivalents are added to C═C bonds. 
     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. 
     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&#39;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 aziridination activity described below. 
     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 25 μM, preferably less than about 5 μM, and even more preferably less than 1 μM. 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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     As used herein, the term “electron withdrawing group” refers to an atom or substituent that has an ability to acquire electron density from an olefin or other atoms or substituents. An “electron withdrawing group” is capable of withdrawing electron density relative to that of hydrogen if the hydrogen atom occupied the same position on the molecule. The term “electron withdrawing group” is well understood by those of skill in the art and is discussed, for example, in Advanced Organic Chemistry by J. March, John Wiley &amp; Sons, New York, N.Y., (1985). Examples of electron withdrawing groups include, but are not limited to, halo (e.g., fluorine, chlorine, bromine, iodine), nitro, carboxy, amido, acyl, cyano, aryl, heteroaryl, —OC(A) 3 , —C(A) 3 , —C(A) 2 -O-C(A′) 3 , —(CO)-Q, —SO 2 —C(A) 3 , —SO 2 -aryl, —C(NQ)Q, —CH═C(Q) 2 , and —C≡C-Q; in which each A and A′ is independently H, halo, —CN, —NO 2 , —OH, or C 1-4  alkyl optionally substituted with 1-3 halo, —OH, or NO 2 ; and Q is selected from H, —OH, and alkyl optionally substituted with 1-3 halo, —OH, —O-alkyl, or —O-cycloalkyl. 
     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 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C 3-5 , C 3-6 , C 4-5 , C 4-6  and C 5- 6 . For example, C 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 also refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl groups can be substituted or unsubstituted. 
     “Alkylene” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated, and linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For instance, a straight chain alkylene can be the bivalent radical of —(CH 2 ) n —, where n is 1, 2, 3, 4, 5 or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene. Alkylene groups can be substituted or unsubstituted. 
     The term “alkoxy” refers to an alkyl group having an oxygen atom that connects the alkyl group to the point of attachment: alkyl-O—. As for alkyl group, alkoxy groups can have any suitable number of carbon atoms, such as C 1-6 . Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. Alkoxy groups can be substituted or unsubstituted. 
     As used herein, the terms “halo” and “halogen” refer to fluorine, chlorine, bromine and iodine. 
     The term “heteroalkyl” refers to an alkyl group of any suitable length and having from 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, —SO— and —SO 2 —. For example, heteroalkyl can include ethers, thioethers and alkyl-amines. The heteroatom portion of the heteroalkyl can be the connecting atom, or be inserted between two carbon atoms. 
     The term “aryl” refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can include any suitable number of 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 substituted or unsubstituted. 
     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 can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, S═O and SO 2  (two double bonded oxygens). 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. The heteroaryl group can include groups such as 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. The heteroaryl groups can also be fused to aromatic ring systems, such as a phenyl ring, to form members including, but not limited to, benzopyrroles such as indole and isoindole, benzopyridines such as quinoline and isoquinoline, benzopyrazine (quinoxaline), benzopyrimidine (quinazoline), benzopyridazines such as phthalazine and cinnoline, benzothiophene, and benzofuran. Other heteroaryl groups include heteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groups can be substituted or unsubstituted. Heteroaryl groups can be linked via any position on the ring. 
     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 3-6 , C 4-6 , C 5-6 , C 3-8 , C 4-8 , C 5-8 , C 6-8 , C 3-9 , C 3-10 , C 3-11 , and C 3-12 . 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), norbornene, and norbornadiene. When cycloalkyl is a saturated monocyclic C 3-8  cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. When cycloalkyl is a saturated monocyclic C 3-6  cycloalkyl, exemplary groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups can be substituted or unsubstituted. 
     The term “iodinane” refers to the chemical substituent 
     
       
         
         
             
             
         
       
     
     where the ‘wavy line’ represents the point of attachment to the remainder of the molecule. 
     Any compound or formula disclosed herein that does not define the chirality of a chiral carbon can be a racemic mixture or may possess an enantiomeric excess of R or S isomers. For example, compounds represented by Formula III, shown below, may possess, 0, 1, or 2 chiral carbons depending on the identities of R 1a , R 1b , and R 2 . Each chiral center may be racemic or may be of a particular enantiomeric excess. A person of skill in the art will also recognize that instances of two chiral carbons within an single aziridine ring can produce two cis and two trans isomers. 
     
       
         
         
             
             
         
       
     
     III. Description of the Embodiments 
     In a first aspect, the invention provides a reaction mixture for producing an aziridination product. The reaction mixture contains an olefinic substrate, a nitrene precursor, and a heme enzyme. 
     In other aspects, the present invention provides heme enzymes including variants thereof that are capable of carrying out the aziridination reactions described herein. Expression vectors and host cells expressing the heme enzymes are also provided by the present invention. 
     In still other aspects, the present invention provides methods for producing an aziridination product. In certain aspects, the present invention provides a method for producing an aziridination product, the method comprising:
         (a) providing an olefinic substrate, a nitrene precursor, and a heme enzyme; and   (b) admixing the components of step (a) in a reaction for a time sufficient to produce an aziridination product.       

     The following sections provide a description of exemplary and preferred embodiments including heme enzymes, expression vectors, host cells, aziridination products such as, e.g., compounds comprising an aziridine functional group, starting materials such as, e.g., olefinic substrates and nitrene precursors, and characteristics and reaction conditions for the in vitro and in vivo aziridination reactions described herein. 
     A. Heme Enzymes 
     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. 
     In certain aspects, the present invention provides compositions comprising one or more heme enzymes that catalyze the conversion of olefinic substrates to aziridination products. In particular embodiments, the present invention provides heme enzyme variants comprising at least one or more amino acid mutations therein that catalyze the formal transfer of a nitrene equivalent to an olefinic substrate, making aziridination products with high stereoselectivity. In preferred embodiments, the heme enzyme variants of the present invention have the ability to catalyze aziridination reactions efficiently, display increased total turnover numbers, and/or demonstrate highly regio- and/or enantioselective product formation compared to the corresponding wild-type enzymes. 
     In some embodiments, the enzyme is a heme enzyme or a variant thereof. 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 aziridination catalysts. 
     In some embodiments, the heme enzyme is a member of one of the enzyme classes set forth in Table A. In other embodiments, the heme enzyme is a variant or homolog of a member of one of the enzyme classes set forth in Table A. 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 A or a fragment thereof (e.g., a truncated heme domain) that is capable of carrying out the aziridination reactions described herein. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 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-ascothate 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-ascothate 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 21-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 
               
               
                   
               
            
           
         
       
     
     In some 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. 
     In certain embodiments, the in vitro methods for producing an aziridination product comprise providing a heme enzyme, variant, or homolog thereof with a reducing agent such as NADPH or a dithionite salt (e.g., Na 2 S 2 O 4 ). In certain other embodiments, the in vivo methods for producing an aziridination product comprise providing whole cells such as  E. coli  cells expressing a heme enzyme, variant, or homolog thereof. 
     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 aziridination 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 aziridination reactions of the present invention. 
     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. 
     In some embodiments, the heme enzyme, variant, or homolog thereof has an enhanced nitrene insertion activity and/or nitrene transfer activity of 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. 
     In some embodiments, the heme enzyme, variant, or homolog thereof has a resting state reduction potential higher than that of NADH or NADPH. 
     In particular embodiments, the heme enzyme comprises a cyctochrome 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 FeIII-protoporphyrin IX cofactor (heme) ligated proximally by a conserved cysteine thiolate (M. T. Green, Current Opinion in Chemical Biology 13, 84 (2009)). 
     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://drnelson.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. 
     In certain embodiments, the cytochrome P450 enzymes of the invention are members of one of the classes shown in Table B (see, http://www.icgeb.org/˜p450srv/P450enzymes.html, the disclosure of which is incorporated herein by reference in its entirety for all purposes). 
     
       
         
           
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 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 7x-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 4  20-monooxygenase 
                 CYP4F 
               
               
                 1.14.13.37 
                 methyltetrahydroprotoberberine 14-monooxygenase 
                 CYP93A1 
               
               
                 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 7x-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 10β-hydroxylase 
                 CYP725A1 
               
               
                 1.14.13.77 
                 taxane 13α-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 11β-monooxygenase 
                 CYP11B 
               
               
                 1.14.15.5 
                 corticosterone 18-monooxygenase 
                 CYP11B 
               
               
                 1.14.15.6 
                 cholesterol monooxygenase (side-chain-cleaving) 
                 CYP11A 
               
               
                 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 17x-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 
               
               
                   
               
            
           
         
       
     
     Table C below lists additional cyctochrome P450 enzymes that are suitable for use in the aziridination reactions of the present invention. The accession numbers in Table C 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 C 
               
             
            
               
                   
               
               
                 Additional cytochrome P450 enzymes of the present invention. 
               
            
           
           
               
               
               
               
            
               
                 Species 
                 Cyp No. 
                 Accession No. 
                 SEQ 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 konkukian 
                 
                 X 
                 YP 037304 
                 30 
               
               
                 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). 
               
            
           
         
       
     
     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 aziridination chemistry without competing side reactions mediated by native P450 catalysis. The invention also provides P450-mediated catalysis that is competent for aziridination 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 aziridination catalysis that includes, but is not limited to, NAD(P)H or sodium dithionite. 
     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). 
     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 aziridination 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. 
     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, thirteen, or all fourteen) of the following amino acid substitutions in SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, T438S, and E442K (SEQ ID NO: 55). 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 (SEQ ID NO: 56). In other instances, the P450 BM3 enzyme variant comprises all fourteen of these amino acid substitutions (i.e., V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, T268A, A290V, L353V, I366V, T438S, and E442K; “BM3-CIS T438S”) in combination with a C400X mutation (e.g., C400S) in SEQ ID NO:1 (SEQ ID NO: 57). In some instances, the P450 BM3 enzyme variant comprises or consists of the heme domain of the BM3-CIS T438S enzyme sequence (e.g., amino acids 1-463 of SEQ ID NO: 1 comprising all fourteen of these amino acid substitutions (SEQ ID NO: 55)). 
     In some embodiments, the P450 BM3 enzyme variant comprises the axial ligand mutation C400S and substitutions to SEQ ID NO:1: V78A, F87V, P142S, T175I, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, A328V, L353V, I366V, L437V, T438S, E442K (SEQ ID NO: 51). In another embodiment, the heme variant comprises the axial ligand mutation C400S and the following amino acid substitutions: L75A, V78A, F87V, P142S, T175I, L181A, A184V, S226R, H236Q, E252G, I263F, T268A, A290V, L353V, I366V, E442K (SEQ ID NO: 53). In some embodiments, the heme enzyme variant is the P-I263F variant (see, Table D). In some embodiments, the heme enzyme variant is the P411 BM3  H2-A-10 I263F (see, Table D). 
     Table D below provides non-limiting examples of cytochrome P450 BM3 variants of the present invention. Each P450 BM3 variant comprises the mutations relative to the wild-type P450 BM3 enzymes as shown. 
     
       
         
           
               
             
               
                 TABLE D 
               
             
            
               
                   
               
               
                 Mutations present in P450 BM3 variants used in the disclosure. 
               
            
           
           
               
               
            
               
                 P450 BM3  variant 
                 Mutations relative to wild-type P450 BM3  (SEQ ID NO: 1) 
               
               
                   
               
               
                 P450 BM3   
                 none 
               
               
                 P450 BM3 -T268A 
                 T268A 
               
               
                 P411 BM3   
                 C400S 
               
               
                 P411 BM3 -T268A 
                 C400S, T268A 
               
               
                 P450 BM3 -CIS 
                 V78A, F87V, P142S, T175I, A184V, S226R, H236Q, 
               
               
                   
                 E252G, T268A, A290V, L353V, I366V, E442K 
               
               
                 P450 BM3 -CIS-T438S 
                 CIS T438S 
               
               
                 P450 BM3 -CIS-T438S C400H 
                 P450 BM3 -CIS T438S, C400H 
               
               
                 P450 BM3 -CIS-T438S C400D 
                 P450 BM3 -CIS T438S, C400D 
               
               
                 P450 BM3 -CIS-T438S C400M 
                 P450 BM3 -CIS T438S, C400M 
               
               
                 P411 BM3 -CIS 
                 P450 BM3 -CIS C400S 
               
               
                 P411 BM3 -CIS-T4385 
                 P450 BM3 -CIS C400S, T438S 
               
               
                 P411 BM3 -CIS-T4385 I263F 
                 P450 BM3 -CIS C400S, T438S, I263F 
               
               
                 (P-I263F) 
                   
               
               
                 P-I263F-A328V 
                 P450 BM3 -CIS C400S, T438S, I263F, A328V 
               
               
                 P-I263F-A328V-L437V 
                 P450 BM3 -CIS C400S, T438S, I263F, A328V, L437V 
               
               
                 P411 BM3 -CIS T438S I263F V87F 
                 P450 BM3 -CIS C400S, T438S I263F, V87F 
               
               
                 P411 BM3 -CIS T438S I263F A268T 
                 P450 BM3 -CIS C400S, T438S I263F, A268T 
               
               
                 P411 BM3 -CIS A268T T438S 
                 P450 BM3 -CIS C400S, A268T, T438S 
               
               
                 P411 BM3  H2-A-10 
                 P450 BM3 -CIS C400S, L75A, L181A 
               
               
                 P411 BM3  H2-A-10 I263F 
                 P450 BM3 -CIS C400S, L75A, L181A, I263F 
               
               
                 P411 BM3  H2-5-F10 
                 P450 BM3 -CIS C400S, L75A, I263A, L437A 
               
               
                 P411 BM3  H2-4-D4 
                 P450 BM3 -CIS C400S, L75A, M177A, L181A, L437A 
               
               
                   
               
            
           
         
       
     
     One skilled in the art will understand that any of the mutations listed in Table D 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. For example, BLAST alignment with the P450 BM3 amino acid sequence as the query sequence can be used to identify the heme axial ligand site and/or the equivalent T268 residue in other cytochrome P450 enzymes. 
     In other aspects, the disclosure provides chimeric heme enzymes such as, e.g., chimeric P450 polypeptides comprised of recombined sequences from P450 BM3 and at least two, or more distantly related P450 enzymes from  Bacillus subtillis  or 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 U.S. Pat. Publ. No. US-2012-0171693-A1 to Arnold et al., the disclosure of which is incorporated herein for all purposes. 
     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. Non-limiting examples of chimeric P450 proteins include those set forth in Table E (C2G9, 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. 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 Chimeric 
                 Heme domain 
                 SEQ ID 
               
               
                   
                 P450s 
                 block sequence 
                 NO 
               
               
                   
                   
               
             
            
               
                   
                 C2G9 
                 22223132 
                 43 
               
               
                   
                 X7 
                 22312333 
                 44 
               
               
                   
                 X7-12  
                 12112333 
                 45 
               
               
                   
                 C2E6 
                 11113311 
                 46 
               
               
                   
                 X7-9 
                 32312333 
                 47 
               
               
                   
                 C2B12  
                 32313233 
                 48 
               
               
                   
                 TSP234 
                 22313333 
                 49 
               
               
                   
                   
               
            
           
         
       
     
     An enzyme&#39;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 aziridination 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 aziridination reactions. 
     When whole cells expressing a heme enzyme are used to carry out an aziridination 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 aziridination reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g cdw   −1 , wherein g cdw  is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol·g cdw   −1 , or from about 1 to about 10 mmol·g cdw   −1 , or from about 5 to about 10 mmol·g cdw   −1 , or from about 0.01 to about 1 mmol·g cdw   −1 , or from about 0.01 to about 0.1 mmol·g cdw   −1 , or from about 0.1 to about 1 mmol·g cdw   −1 , or greater than 1 mmol·g cdw   −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·g cdw   −1 . 
     When whole cells expressing a heme enzyme are used to carry out a aziridination 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 cdw   −1  h −1 ). In general, in vivo aziridination reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·g cdw   −1  h −1 , wherein g 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 cdw   −1  h −1 , or from about 0.1 to about 0.5 g g cdw   −1  h −1 , or greater than 0.5 g g cdw   −1  h −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 cdw   −1  h −1 . 
     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. Aziridination activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein. 
     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. 
     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. 
     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). 
     Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 or pET22b(+) (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. 
     The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell. 
     Suitable bacterial host cells include, but are not limited to, BL21  E. coli , DE3 strain  E. coli, E. coli  M15, DH5α, DH10β, 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, NS0 hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell. 
     In certain embodiments, the present invention provides heme enzymes such as the P450 variants described herein that are active aziridination 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 aziridination reactions of the present invention. In some embodiments, whole cell catalysts containing a P450 enzymes variant described herein significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes. 
     B. Compounds 
     The methods of the invention can be used to provide a number of aziridination products. The aziridination products described herein can be useful starting materials or intermediates for the synthesis of compounds. 
     The olefinic substrates useful in the present invention are represented by a structure of Formula I: 
     
       
         
         
             
             
         
       
     
     For compounds of Formula I, R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, C 1-8 heteroalkyl, aryl, heteroaryl, C 1-12 cycloalkyl, C 3-10 heterocyclyl, —Y 1 -aryl, —Y 1 -heteroaryl, —Y 1 —C 1-12 cycloalkyl and —Y 1 —C 3-10 heterocyclyl; Y 1  is C 1-8 alkylene; each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo, cyano, and halogen. In some embodiments, each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 8 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S. 
     In certain instances, R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, aryl, heteroaryl, C 1-12 cycloalkyl, and C 3-10 heterocyclyl, each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy, and halogen. 
     In some embodiments, R 1a  is a substituted phenyl group or a naphthalenyl, wherein the phenyl group is substituted with 1 to 2 a methyl, chloro, or C 1 alkyoxy groups. 
     In some embodiments, R 1b  is H or methyl. 
     In some embodiments, R 2  is H or methyl. In some embodiments, R 2  is H. 
     The nitrene precursors useful in the present invention have a structure according to the Formula IIa or IIb: 
     
       
         
         
             
             
         
       
     
     wherein:
         R 3  is selected from the group consisting of C 1-18  alkyl, C 1-8 heteroalkyl, C 3-12 cycloalkyl, aryl, heteroaryl, C 3-10 heterocyclyl, —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ; X 1  is independently selected from the group consisting of H and sodium, and X 2  is independently selected from the group consisting of halogen, —SO 2 R a , —CO 2 R b , —PO 3 R b R c , optionally X 1  and X 2  can be taken together to form iodinane; R a  is independently selected from the group consisting of C 1-8 alkyl, hydroxy, C 1-8 alkoxy, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy. In some embodiments, each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S.       

     In certain instances, R 3  is selected from the group consisting of aryl, —SO 2 R a , —COR a , —CO 2 R b , and —PO 3 R b R c ; X 1  is independently selected from the group consisting of H and sodium, and X 2  is independently selected from the group consisting of halogen, —SO 2 R a , optionally X 1  and X 2  can be taken together to form iodinane; R a  is independently selected from the group consisting of C 1-8 alkyl, C 1-8 alkoxy, and aryl; R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, and aryl; wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, and halogen. In some embodiments, each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S. 
     In some embodiments, the nitrene precursor has a structure selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     In some instances, the nitrene precursor is 
     
       
         
         
             
             
         
       
     
     In some embodiments, the aziridination product is a compound according to Formula III: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, C 1-8 heteroalkyl, aryl, heteroaryl, C 1-12 cycloalkyl, C 3-10 heterocyclyl, —Y 1 -aryl, —Y 1 -heteroaryl, —Y 1 —C 1-12 cycloalkyl and —Y 1 —C 3-10 heterocyclyl; Y 1  is C 1-8 alkylene; each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo, cyano, and halogen; R 3  is selected from the group consisting of C 1-18  alkyl, C 1-8 heteroalkyl, C 3-12 cycloalkyl, aryl, heteroaryl, C 3-10 heterocyclyl, —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ; R a  is independently selected from the group consisting of C 1-8 alkyl, hydroxy, C 1-8 alkoxy, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl, wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy. In some embodiments, each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S. 
           
         
       
    
     In some instances, R 1a  and R 1b  are independently selected from the group consisting of H, C 1-8 alkyl, aryl, heteroaryl, C 1-12 cycloalkyl, and C 3-10 heterocyclyl; R 2  is selected from the group consisting of H and C 1-8  alkyl; each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 3 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy, and halogen; and R 3  is selected from the group consisting of —SO 2 R a , —COR a , —CO 2 R b , and —PO 3 R b R c ; R a  is independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; R b  and R c  are independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl, wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-2 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy. 
     In some embodiments, the aziridination product is a compound according to Formula IIIa: 
     
       
         
         
             
             
         
       
         
         
           
             wherein R 1a , R 1b , and R 2  are independently selected from the group consisting of H, C 1-18 alkyl, C 1-8 heteroalkyl, aryl, heteroaryl, C 1-12 cycloalkyl, C 3-10 heterocyclyl, —Y 1 -aryl, —Y 1 -heteroaryl, —Y 1 —C 1-12 cycloalkyl and —Y 1 —C 3-10 heterocyclyl; Y 1  is C 1-8 alkylene; each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 5 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy hydroxyl, amino, thiol, carboxy, amido, oxo, thioxo, cyano, and halogen; R 3  is selected from the group consisting of C 1-18  alkyl, C 1-8 heteroalkyl, C 3-12 cycloalkyl, aryl, heteroaryl, C 3-10 heterocyclyl, —SO 2 R a , —COR a , —CO 2 R b , —PO 3 R b R c , and —CONR b R c ; R a  is independently selected from the group consisting of C 1-8 alkyl, hydroxy, C 1-8 alkoxy, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; R b  and R c  are independently selected from the group consisting of C 1-8 alkyl, C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl, wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-5 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy. In some embodiments, each aryl contains between 6-14 carbon atoms, each heteroaryl group has from 5 to 10 ring atoms and from 1-3 heteroatoms selected from N, O and S, and each heterocyclyl group has from 1-3 heteroatoms selected from N, O and S. 
           
         
       
    
     In some instances, R 1a  and R 1b  are independently selected from the group consisting of H, C 1-8 alkyl, aryl, heteroaryl, C 1-12 cycloalkyl, and C 3-10 heterocyclyl; R 2  is selected from the group consisting of H and C 1-8  alkyl; each R 1a , R 1b , and R 2  is optionally substituted with from 1 to 3 substituents independently selected from the group consisting of C 1-3 alkyl, alkoxy, and halogen; and R 3  is selected from the group consisting of —SO 2 R a , —COR a , —CO 2 R b , and —PO 3 R b R c ; R a  is independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl; R b  and R c  are independently selected from the group consisting of C 3-12 cycloalkyl, aryl, heteroaryl, and C 3-8 heterocyclyl, wherein within each R 3 , R a , R b , and R c  can be optionally substituted with from 1-2 R d  substituents; and each R d  is independently selected from the group consisting of C 1-3 alkyl, halogen, and hydroxy. 
     In some embodiments, compounds of Formula IIIa are further reaction products of an aziridine ring that has been opened after attack from a nucleophile, such as a hydroxyl group. Compounds of formula IIIa can be produced when the aziridination reactions described herein are performed under aqueous reaction conditions. 
     In some embodiments, the aziridination product has a structure selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     One of skill in the art will appreciate that stereochemical configuration of certain of the products herein will be determined in part by the orientation of the product of the enzymatic step. Certain of the products herein will be “cis” compounds or “Z” compounds. Other products will be “trans” compounds or “E” compounds. One product where cis or trans orientations are possible is the formation of an aziridine ring. The cis configuration of an aziridine ring is when the highest priority substituents are on the same side of the ring (e.g., Formula III when R 1a  and R 2  are the highest priority substituents and on the same side of the aziridine ring), while the trans configuration of an aziridine ring is when the highest priority substituents are on the opposite side of the ring. 
     In certain instances, two cis isomers and two trans isomers can arise from the reaction of an olefin substrate and a nitrene precursor. 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 product—that is, whether a given chiral center exhibits the right-handed “R” configuration or the left-handed “S” configuration—will depend on factors including the structures of the particular substrate and nitrene precursor used in the reaction, as well as the identity of the enzyme. The relative stereochemistry—that is, whether a product exhibits a cis or trans configuration—as well as for the distribution of product mixtures will also depend on such factors. 
     In certain instances, the 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. 
     The distribution of 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. Taking the reaction scheme in  FIG. 8  as a non-limiting example, for instance, the enantiomeric excess of the “S” enantiomer can be calculated using the formula: % ee S =[(χ S −χ R )/(χ S +χ R )]×100%, wherein χ is the mole fraction for a given enantiomer. The enantiomeric excess of the “R” enantiomer (% ee R ) can be calculated in the same manner. In less otherwise specified, % ee is reported as % ee S . 
     In general, 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 S  values or % ee R  values. 
     Accordingly, some embodiments of the invention provide methods for producing a plurality of aziridination products having a % ee S  of from about −90% to about 90%. In some embodiments, the % ee S  is at least 90%. In some embodiments, the % ee S  is at least −99%. In some embodiments, the % ee R  is from about −90% to about 90%. In some embodiments, the % ee R  is at least 90%. In some embodiments, the % ee R  is at least −99%. 
     The methods of the disclosure can also be assessed in terms of the diastereoselectivity and/or enantioselectivity of the aziridination reaction—that 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. The diastereoselectivity and/or enantioselectivity of an aziridination reaction is dependent on a number of factors including the olefinic substrate, nitrene precursor, and heme enzyme used. 
     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%&gt;to about 80%&gt;diastereoselective, or from about 40%&gt;to about 60%) diastereoselective, or from about 1% to about 25% diastereoselective, or from about 25% o 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 disclosure 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. 
     C. Reaction Conditions 
     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, aziridination reaction mixtures contain from about 0.01 mol % to about 10 mol % heme enzyme with respect to the nitrene precursor 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. 
     The concentration of olefinic substrate and nitrene precursor are typically in the range of from about 100 μM to about 1 M. The concentration can be, for example, from about 100 μM 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 μM to about 500 mM, 500 μM 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 nitrene precursor can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. The concentration of olefinic substrate or nitrene precursor can be about 1, 2.5, 5, 7.5, 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. 
     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 2 , and salts of Mn 2+  and Mg 2+ ), denaturants (e.g., urea and guandinium 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), 3-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 μM 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 μM, or about 10 μM, or about 100 μM, 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 nitrene precursor. 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). 
     Reactions are conducted under conditions sufficient to catalyze the formation of an aziridination 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 aziridination reaction occurs in the aqueous phase. In some embodiments, the heme enzymes 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 nitrene precursor. 
     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). Aziridination 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 aziridination reactions. Other densities can be useful, depending on the cell type, specific heme enzymes, or other factors. 
     IV. Examples 
     The present 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 
     Aziridination Activity of Cytochrome P450 Variants and Other Heme Proteins 
     This example illustrates the aziridination activity of known cytochrome P450 variants and other heme containing enzymes. 
     Previous studies have shown that cytochrome P450 and mutants thereof can catalyze a wide variety of chemical reactions including cyclopropanation, sulfinde imidation, and C—H amination. In order to assess the potential of a cytochrome P450 or a mutant thereof to catalyze an aziridination reaction, engineered variants of cytochrome P450 BM3  and P411 BM3 -CIS-T438S, previously found to be effective for intramolecular C—H amination and sulfide imidation, were tested for aziridination activity. Cytochrome P450 BM3  is a naturally occurring enzyme found in the soil bacterium  bacillus megaterium , and P411 BM3 -CIS-T438S is a 14 mutation variant of P450 BM3  (see Table 2 for mutations from wild-type P450 BM3 ). P411 BM3 -CIS-T438S is called a “P411” due to the change in the characteristic CO-bound Soret peak from 450 to 411 nm effected by mutation of the cysteine residue that coordinates the heme iron to serine (C400S). This axial cysteine is completely conserved in cytochrome P450s and is required for the native monooxygenase activity. Thus, the P411 enzyme is no longer a “cytochrome P450”, nor does it exhibit its native hydroxylation activity. However, the C400S mutation increases the non-natural carbene transfer activities of P450 BM3  and other P450s. Two crystal structures of P411 variants of P450 BM3  show that S400 coordinates the iron and causes no significant structural perturbation in the substrate binding pocket. 
     The aziridination activity of P411 BM3 -CIS-T438S was tested using styrene derivatives as the olefin substrate and tosyl azide (TsN 3 ) as the nitrene precursor (Table 1). Tosyl azide was completely consumed in this reaction, the major product of which was the azide reduction product p-toluenesulfonamide (&gt;300 total turnovers (TTN), not shown in Table 1). Amidoalcohol 2 appeared as a minor product. Control experiments showed that the desired aziridine product rapidly decomposes under aqueous reaction conditions to the corresponding amidoalcohol 2 ( FIGS. 2A-B ,  3 A-D, and  4 A-D). Degradation of this aziridine product has also been observed in studies with small-molecule catalysts (Ando, T.; et al.,  Tetrahedron  54, 13485-13494 (1998) and Kiyokawa, K. et al.,  Org. Lett.,  15, 4858-4861 (2013)). It was thus inferred that production of 2 is directly related to the nitrene transfer activity of the enzyme toward olefin 1. 
                     TABLE 1                  Total turnovers (TTN) to product for aziridination catalyzed by purified holoenzymes       P411 BM3 -CIS-T438S (P) and P411 BM3 -CIS-T438S-I263F (P-I263F) with selected styrenyl       olefins 1, 3, and 5 and tosyl azide. a                                                                 Enzyme   TTN 2  b     TTN 4   TTN 6                                     P411 BM3 -CIS-T438S (P)   15   8   5       P-I263F   150   160   190                 a Reactions were performed in 0.1 M KPi buffer pH = 8.0 using 0.2 mol % enzyme and NADPH as reductant, with 2.5 mM tosyl azide and 7.5 mM olefin. Detailed reaction conditions can be found in the supporting information.              b  TTN=Total turnover number. TTNs were determined by HPLC analysis.
 
     This low level of nitrene transfer activity to 4-methoxystyrene olefin of the P411 BM3 -CIS-T438S enzyme prompted investigation of other variants. A small set of cytochrome P450 BM3  variants and heme proteins prepared for other studies were chosen in order to assess how changes in the protein sequence affect nitrene transfer to olefin substrates. Table 2 shows the variants of the cytochrome P450 BM3  mutants tested, and Tables 3 and 4 illustrate the results of these tests. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Mutations present in P450 BM3 variants tested. 
               
            
           
           
               
               
            
               
                 Enzyme 
                 Mutations relative to wild-type P450 BM3   
               
               
                   
               
               
                 P450 BM3   
                 none 
               
               
                 P450 BM3 -CIS 
                 V78A, F87V, P142S, T175I, A184V, S226R, H236Q, 
               
               
                   
                 E252G, T268A, A290V, L353V, I366V, E442K 
               
               
                 P450 BM3 -CIS T438S 
                 P450 BM3 -CIS T438S 
               
               
                 P450 BM3 -CIS T438S C400H 
                 P450 BM3 -CIS T438S, C400H 
               
               
                 P450 BM3 -CIS T438S C400D 
                 P450 BM3 -CIS T438S, C400D 
               
               
                 P450 BM3 -CIS T438S C400M 
                 P450 BM3 -CIS T438S, C400M 
               
               
                 P411 BM3 -CIS 
                 P450 BM3 -CIS C400S 
               
               
                 P411 BM3 -CIS T438S 
                 P450 BM3 -CIS T438S, C400S 
               
               
                 P411 BM3 -CIS A268T T438S 
                 P450 BM3 -CIS A268T, C400S, T438S 
               
               
                 P411 BM3 -H2-5-F10 
                 P450 BM3 -CIS L75A, I263A, C400S, L437A 
               
               
                 P411 BM3 -H2-A-10 
                 P450 BM3 -CIS L75A, L181A, C400S 
               
               
                 P411 BM3 -H2-4-D4 
                 P450 BM3 -CIS L75A, M177A, L181A, C400S, L437A 
               
               
                 P411 BM3   
                 P450 BM3 -C400S 
               
               
                 P450 BM3 -T268A 
                 T268A 
               
               
                 P411 BM3 -T268A 
                 P450 BM3 -T268A, C400S 
               
               
                 P411 BM3 -CIS T438S I263F 
                 P450 BM3 -CIS T438S, I263F, C400S 
               
               
                 (P-I263F) 
                   
               
               
                 P411 BM3 -CIS T438S I263F 
                 P450 BM3 -CIS T438S, I263F, C400S, V87F 
               
               
                 V87F 
                   
               
               
                 P411 BM3 -CIS T438S I263F 
                 P450 BM3 -CIS T438S, I263F, C400S, A268T 
               
               
                 A268T 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Panel of P450 BM3  purified enzymes tested for aziridination reactivity with 4-methoxystyrene and tosyl azide. a   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Entry 
                 Enzyme 
                 TTN 2 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 P411 BM3 -CIS T438S (P) 
                 15 
               
               
                 2 
                 P450 BM3 -CIS T438S 
                 &lt;1 
               
               
                 3 
                 P450 BM3 -CIS T438S C400H 
                 3 
               
               
                 4 
                 P450 BM3 -CIS T438S C400D 
                 4 
               
               
                 5 
                 P450 BM3 -CIS T438S C400M 
                 4 
               
               
                 6 
                 P411 BM3 -CIS A268T T438S 
                 &lt;1 
               
               
                 7 
                 P411 BM3 -H2-5-F10 
                 8 
               
               
                 8 
                 P411 BM3 -H2-A-10 
                 4 
               
               
                 9 
                 P411 BM3 -H2-4-D4 
                 5 
               
               
                 10 
                 P450 BM3   
                 &lt;1 
               
               
                 11 
                 P411 BM3   
                 3 
               
               
                 12 
                 P450 BM3 -T268A 
                 2 
               
               
                 13 
                 P411 BM3 -T268A 
                 4 
               
               
                 14 
                 P411 BM3 -CIS T438S I263F (P-I263F) 
                 150 
               
               
                 14 
                 P411 BM3 -CIS T438S I263F V87F 
                 19 
               
               
                 15 
                 P411 BM3 -CIS T438S I263F A268T 
                 &lt;1 
               
               
                   
               
               
                   a “P411” denotes Ser-mutated (C400S) variant of cytochrome P450 BM3 . Variant IDs and specific amino acid substitutions in each can be found in Table 2. TTN—total turnover number. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Heme and other heme-containing proteins tested for activity in the above reaction 
               
               
                 (Table 3) with 4-methoxystyrene. Myoglobin and cytochrome c were purchased as lyophilized 
               
               
                 powder from Sigma Aldrich. P450Rhf mutants were expressed and purified as described in the 
               
               
                 methods section; P450CYP119 was expressed and purified as described in Heel, T. et al., 
               
               
                   ChemBioChem ., 15, 2556(2014). 
               
            
           
           
               
               
               
            
               
                 Entry 
                 Catalyst 
                 TTN 2 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Hemin 
                 &lt;1 
               
               
                 2 
                 Hemin + BSA 
                 &lt;1 
               
               
                 3 
                 Myoglobin (horse heart) 
                 &lt;1 
               
               
                 4 
                 Oytochrome c (bovine heart) 
                 &lt;1 
               
               
                 5 
                 CYP119 C317S 
                 7 
               
               
                 6 
                 CYP119 T213A C317H 
                 &lt;1 
               
               
                 7 
                 P450 Rhf   
                 &lt;1 
               
               
                   
               
            
           
         
       
     
     P450 BM3  sequences lacking the C400S and/or T268A mutations were not active, nor did the Fe(II)-protoporphyrin IX (PPIX) cofactor catalyze aziridination under these conditions. Mutants differing from P411 BM3 -CIS-T438S by 2-5 alanine mutations in the active site showed some aziridination activity (4-8 TTN), but none was more productive than P411 BM3 -CIST438S. A set of enzymes containing different axial mutations were tested, including the S400H, S400D, and S400M mutants of P411 BM3 -CIS-T438S. These enzymes were also only weakly active, giving 2 at levels lower than P411 BM3 -CIS-T438S (3-4 TTN). Myoglobin (horse heart), cytochrome c (bovine heart), and cytochrome P450 Rhf  (from  Rhodococcus  sp. NCIMB 9784) were all inactive for this intermolecular aziridination (Table 4). An engineered variant of the thermostable cytochrome P450 from  Sulfolobus acidocaldarius , CYP119, that contained an axial cysteine-to-serine mutation (C317S) did catalyze low levels of aziridination (˜7 TTN). This demonstrates that mutations previously described to activate non-natural nitrene-transfer activity in P450 BM3  can confer measurable activity on other P450s as well. 
     Of all the enzymes tested, a variant of P411BM3-CIS-T438S having a single active-site substitution, I263F, was the most active toward 4-methoxystyrene, providing 150 total turnovers in the formation of amido-alcohol 2 from 4-methoxystyrene (Table 3). P-I263F was even more productive when the reactions were carried out using whole  Escherichia coli  cells expressing this enzyme ( FIG. 5 ), consistent with our previous observations that enzyme-catalyzed metal-nitrenoid and metal-carbenoid transfer activities improved when the reactions were performed with whole cells. No aziridine product was observed when cells not expressing the P411 catalyst were used. 
     Example 2 
     Optimizing Cytochrome P450 Aziridination Activity 
     This example illustrates bacterial cytochrome P450s that are engineered to catalyze highly stereoselective nitrene transfers to olefin substrates to make aziridines. 
     The P-I263F enzyme identified in the initial studies of enzyme catalyzed aziridination provided enough aziridine product in whole-cell reactions to allow for screening variants in 96-well plate format. Thus, further improvement of aziridination productivity was sought by mutagenesis of this enzyme and screening for aziridination productivity. Site-saturation mutagenesis (SSM) libraries were created at several active site positions that were previously shown to influence productivity and enantioselectivity in other non-natural reactions (A78, L181, T438, A328). Screening of these single SSM libraries for aziridination of 4-methylstyrene (3) identified P-I263F-A328V, with slightly improved yield and substantially improved % ee (96% ee S ; entry 4, Table 5). Another round of SSM performed on this variant at additional active site positions (F87, T268, L437) resulted in P-I263F-A328V-L437V with improved aziridine yield and a further increase in enantioselectivity (99% ee S ). The P-I263F-L437V and P-I263F-A328V mutants were both less selective than P-I263F-A328V-L437V, demonstrating that both new mutations contribute to the very high enantioselectivity. Importantly, the yield of sulfonamide side product 7 diminished over the course of active site evolution, to the extent that aziridine 4 became the major product of the reaction catalyzed by P-I263F-A328V-L437V. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Improvement in yield and % ee for aziridine product 4 with active-site evolution of P411 BM3 CIS-T438S (P). a   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Entry 
                 Enzyme 
                 % yield 4 
                 % yield 7 
                 % ee 4 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 No enzyme 
                 0 
                 95 
                 nd 
               
               
                 2 
                 P411 BM3 -CIS-T438S 
                 1.1 
                 95 
                 25 
               
               
                 3 
                 P-I263F 
                 40 
                 54 
                 55 
               
               
                 4 
                 P-I263F-A328V 
                 43 
                 50 
                 96 
               
               
                 5 
                 P-I263F-L437V 
                 37 
                 52 
                 95 
               
               
                 6 
                 P-I263F-A328V-L437V 
                 55 
                 43 
                 99 
               
               
                   
               
               
                   a Reactions were carried out using whole  E. coli  cells resuspended in M9-N reaction buffer under anaerobic conditions, with 2.5 mM tosyl azide and 7.5 mM 4-methylstyrene. Yield is based on tosyl azide. See methods for detailed reaction set up and quantification procedures. 
               
               
                   b % ee determined by SFC analysis and calculated as (S − R) / (S + R). 
               
               
                   c ‘No enzyme’ reactions were carried out using whole cells with no P411 enzyme expressed, as described in the SI methods 
               
            
           
         
       
     
     Because the azide is fully consumed in these reactions, the improved aziridine yield could result from either an increase in the rate of aziridine formation or a decrease in the rate of competing azide reduction, or from a combination of both. To address this, initial rates of reaction were measured with the PI263F, P-I263F-A328V, and P-I263F-A328V-L437V enzymes as purified holoenzymes ( FIGS. 6 and 7A -C). Initial rates of aziridination for the purified enzymes reflected the yield improvements observed in whole cells: P-I263F and P-I263FA328V have similar turnover frequencies (15-16 min −1 ), while P-I263F-A328V-L437V, having both new mutations, was improved (TOF ˜24 min −1 ). The initial turnover frequency of sulfonamide formation in vitro was similar for all the enzymes, and faster than aziridine formation (TOFs ˜26-29 min −1 . 
     Example 3 
     Productivity and Enantioselectivity of Select Cytochrome P450 Enzymes 
     This example illustrates the aziridination productivity and enantioselectivity of P-I263F-A328V-L437V when reacted with different substrates. This example also illustrates the aziridination productivity and enantioselectivity using enzyme variant P411 BM3  H2-A-10 I263F. 
     Having obtained a variant capable of high productivity and enantioselectivity for the aziridination of 4-methylstyrene (3), whole-cell reactions with different substituted styrene substrates were investigated (Table 6). No correlation between the electronics of the aryl substituent and the productivity of the enzyme were observed. In general, the evolved enzyme was more productive with styrenes substituted at the 4-position, though the highest productivity was observed with styrene itself. The evolved enzyme provided 600 catalytic turnovers for the formation of aziridine 6, corresponding to a 70% yield of 6 (entry 3 in Table 6). With higher styrene and tosyl azide loading, the enzyme catalyzed 1,000 turnovers for aziridination, while retaining high (S)-selectivity (99% ee) ( FIG. 8 ). Both 3-methylstyrene and 3-chlorostyrene were significantly less reactive than their 4-substituted counterparts, giving 85 and 21 turnovers, respectively, compared to 450 and 290 turnovers (entries 2, 4, 5, 6 in Table 6). The evolved enzyme is an exceptionally enantioselective aziridination catalyst with styrene entries 2-4 (Table 6), giving 99% ee in favor of the (S)-enantiomer with these three substrates. Both 4-methoxystyrene and α-methylstyrene (entries 1 and 8 in Table 6) gave exclusively racemic amido-alcohol product. Formation of the amido-alcohol product from these substrates may result from carbocation stabilization at the benzylic position due to the resonance and hyperconjugative stabilization provided by the respective p-OMe and α-Me groups, leading to decomposition of the aziridine product and subsequent carbocation quenching with water. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Substrate aziridination with P-I263F-A328V-L437V showing productivity in terms of 
               
               
                 TTN and selectivity in % ee for each product. a   
               
            
           
           
               
               
               
               
               
               
            
               
                 Entry 
                 Olefin 
                 Product 
                 TTN 
                 % yield 
                 % ee  b   
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 390 
                 47 
                 rac 
               
               
                   
               
               
                 2 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 450 
                 55 
                 99 
               
               
                   
               
               
                 3 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 600 
                 70 
                 99 
               
               
                   
               
               
                 4 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 290 
                 36 
                 99 
               
               
                   
               
               
                 5 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 21 
                 2 
                 95 
               
               
                   
               
               
                 6 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 85 
                 10 
                 95 
               
               
                   
               
               
                 7 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 130 
                 15 
                 81 
               
               
                   
               
               
                 8 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 83 
                 10 
                 rac 
               
               
                   
               
               
                 9 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 53 
                 6 
                 88 
               
               
                   
               
               
                   a Reactions were carried out with whole cells expressing P-I263F-A328V-L437V under anaerobic conditions, with 2.5 mM tosyl azide and 7.5 mM olefin. Reactions were allowed to proceed for 4 hours at room temperature. 
               
               
                   b  % ee determined as (S − R) / (S + R). Absolute configurations were assigned based on analogy to 6. rac = racemic. 
               
            
           
         
       
     
     Although previous work has highlighted the importance of modulating heme electronic properties to access non-natural reactivity (McIntosh, J. A.; et al.,  Angew. Chem., Int. Ed.  52, 9309-9312 (2013); Hyster, T. K.; et al.,  J. Am. Chem. Soc.,  136, 15505-15508 (2014); Coelho, P. S.; et al.,  Nat. Chem. Biol.  9, 485-487 (2013)), here it was observed that strong gains in aziridination activity are brought about by mutations on the distal heme side, suggesting that their effect may be the result of improving substrate binding and orientation, a hallmark of enzyme catalysis that is notable for a new-to-nature reaction such as P450-catalyzed nitrene transfer. 
     P-I263F-A328V-L437V is an exceptionally (S)-selective aziridination catalyst with olefin entries 2-4 (Table 6), giving 99% ee in favor of the (S)-enantiomer with these three substrates. Also identified in this work is the P411 BM3  H2-A-10 I263F enzyme variant which is an I263F mutant of the P411 BM3  H2-A-10 enzyme identified in a previous study. The P411 BM3  H2-A-10 I263F enzyme is able to catalyze the aziridination reaction with enantioselectivity that favors the R-enantiomer (84% ee in favor of (R)-enantiomer, see reaction scheme below). 
     
       
         
         
             
             
         
       
     
     Example 4 
     Synthesis of Substrates and Standards 
     The following example illustrates the synthesis of substrates and standards. 
     N-(2-hydroxy-2-(4-methoxyphenyl)ethyl)-4-methylbenzenesulfonamide (2) 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Srinivas, B. et al.,  J. Mol. Catal. A: Chem.,  261, 1-5 (2007). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.72 (d, 2H, J=8.1 Hz), 7.29 (d, 2H, J=8.3 Hz), 7.19 (d, 2H, J=8.6 Hz), 6.84 (d, 2H, 8.6 Hz), 5.06 (dd, 1H, J=8.1, 4.6 Hz), 4.73 (dd, 1H, J=8.7, 3.7 Hz), 3.78 (s, 3H), 3.20 (ddd, 1H, J=13.3, 8.1, 3.7 Hz), 3.01 (ddd, 1H, J=13.2, 8.6, 4.6 Hz), 2.42 (s, 3H) 
       13 C NMR (101 MHz, CDCl 3 ): δ 159.66, 143.69, 136.86, 133.00, 129.90, 127.26, 127.21, 114.16, 72.50, 55.44, 50.30, 21.66 
     HRMS (FAB+): calculated for C 16 H 18 NO 4 S ([M+H]+): 320.0956. found: 320.0950. 
     N-(p-Tolylsulfonyl)-2-(p-methylphenyl)aziridine (4) 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Huang, C. Y. et al.,  J. Am. Chem. Soc.,  134, 9541-9544 (2012); Kiyokawa, K. et al.,  Org. Lett.  15, 4858-4861 (2013); Evans. D. et al.,  J. Am. Chem. Soc.,  116, 2742-2753 (1994)). 
       1 H NMR (300 MHz, CDCl 3 ): δ 7.86 (d, 2H, J=8.3 Hz), 7.32 (d, 2H, J=8.3 Hz), 7.10 (s, 4H), 3.74 (dd, 1H, J=7.2, 4.5 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.43 (s, 3H), 2.38 (d, 1H, J=4.5 Hz), 2.31 (s, 3H). 
     N-(p-Tolylsulfonyl)-2-phenylaziridine (6) 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Huang, C. Y. et al.,  J. Am. Chem. Soc.,  134, 9541-9544 (2012); Kiyokawa, K. et al.,  Org. Lett.  15, 4858-4861 (2013); Evans. D. et al.,  J. Am. Chem. Soc.,  116, 2742-2753 (1994)). 
       1 H NMR (300 MHz, CDCl 3 ): δ 7.87 (d, 2H, J=8.3 Hz), 7.19-7.36 (m, 7H), 3.77 (dd, 1H, J=7.2, 4.5 Hz), 2.98 (d, 1H, J=7.2 Hz), 2.43 (s, 3H), 2.39 (d, 1H, J=4.5 Hz) 
     N-(p-Tolylsulfonyl)-2-(p-methoxyphenyl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Huang, C. Y. et al.,  J. Am. Chem. Soc.,  134, 9541-9544 (2012); Kiyokawa, K. et al.,  Org. Lett.  15, 4858-4861 (2013); Evans. D. et al.,  J. Am. Chem. Soc.,  116, 2742-2753 (1994)). 
       1 H NMR (500 MHz, CDCl 3 ): δ 7.87 (d, 2H, J=8.3 Hz), 7.34 (d, 2H, J=8.5 Hz), 7.14 (d, J=8.7 Hz, 2H), 6.83 (d, J=8.7, 2H), 3.78 (s, 3H), 3.75 (dd, 1H, J=7.2, 4.5 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.39 (d, 1H, J=4.5 Hz) 
     N-(p-Tolylsulfonyl)-2-(p-chlorophenyl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Huang, C. Y. et al.,  J. Am. Chem. Soc.,  134, 9541-9544 (2012); Kiyokawa, K. et al.,  Org. Lett.  15, 4858-4861 (2013); Evans. D. et al.,  J. Am. Chem. Soc.,  116, 2742-2753 (1994)). 
       1 H NMR (300 MHz, CDCl 3 ): δ 7.86 (d, 2H, J=8.3 Hz), 7.34 (d, 2H, J=7.9 Hz), 7.26 (d, 2H, J=8.5 Hz), 7.15 (d, 2H, J=8.5 Hz), 3.73 (dd, 1H, J=7.2, 4.4 Hz), 2.98 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.34 (d, 1H, J=4.4 Hz) 
     N-(p-Tolylsulfonyl)-2-(m-chlorophenyl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Craig II, R. A.; et al.  Chem. Eur. J.,  20, 4806-4813 (2014)). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.87 (d, 2H, J=8.3 Hz), 7.35 (d, 2H, J=7.7 Hz), 7.19-7.26 (m, 3H), 7.12 (dt, 1H, J=6.8, 1.8 Hz), 3.73 (dd, 1H, J=7.2, 4.3 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.35 (d, 1H, J=4.4 Hz) 
     N-(p-Tolylsulfonyl)-2-(m-methylphenyl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Gao, G. Y. et al.,  Org. Lett.,  7, 3191-3193 (2005)). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.87 (d, 2H, J=8.3 Hz), 7.33 (d, 2H, J=8.6 Hz), 7.01-7.20 (m, 4H), 3.74 (dd, 1H, J=7.2, 4.5 Hz), 2.96 (d, 1H, J=7.2 Hz), 2.43 (s, 3H), 2.38 (d, 1H, J=4.5 Hz), 2.30 (s, 3H) 
     N-(p-Tolylsulfonyl)-2-(2,4-dimethylphenyl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.90 (d, 2H, J=8.4 Hz), 7.34 (d, 2H, J=8.5 Hz), 6.91-7.00 (m, 3H), 3.84 (dd, 1H, J=7.2, 4.6 Hz), 2.97 (d, 1H, J=7.2 Hz), 2.44 (s, 3H), 2.35 (s, 3H), 2.32 (d, 1H, J=4.6 Hz), 2.28 (s, 3H) 
       13 C NMR (101 MHz, CDCl 3 ): δ 144.72, 137.95, 136.72, 135.15, 130.89, 130.32, 129.84, 128.11, 126.82, 125.98, 39.61, 35.07, 21.75, 21.11, 19.08 
     HRMS (FAB+): calculated for C 17 H 20 NO 2 S ([M+H]+): 302.1215. found: 302.1210. 
     N-(2-hydroxy-2-phenylpropyl)-4-methylbenzenesulfonamide 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Srinivas, B. et al.,  J. Mol. Catal. A: Chem.,  261, 1-5 (2007). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.67 (d, 2H, J=8.3 Hz), 7.24-7.38 (m, 7H), 4.59 (s, 1H), 3.22 (dd, 1H, J=12.8, 8.5 Hz), 3.12 (dd, 1H, J=12.8, 4.8 Hz), 2.42 (s, 3H), 1.56 (s, 3H) 
       13 C NMR (101 MHz, CDCl 3 ): δ 144.87, 143.73, 136.73, 129.93, 128.75, 127.60, 127.19, 124.93, 73.81, 53.99, 27.62, 21.68 
     HRMS (FAB+): calculated for C 16 H 20 NO 3 S ([M+H]+): 306.1164. found: 306.1160. 
     N-(p-Tolylsulfonyl)-2-(naphthalene-2-yl)aziridine 
     
       
         
         
             
             
         
       
     
     Synthesized as previously reported in Ando, T et al.,  Tetrahedron,  54, 13485-13494 (1998) with spectral data in agreement with literature reported values (Huang, C. Y. et al.,  J. Am. Chem. Soc.,  134, 9541-9544 (2012); Kiyokawa, K. et al.,  Org. Lett.  15, 4858-4861 (2013); Evans. D. et al.,  J. Am. Chem. Soc.,  116, 2742-2753 (1994)). 
       1 H NMR (400 MHz, CDCl 3 ): δ 7.90 (d, 2H, J=8.3 Hz), 7.75-7.81 (m, 3H), 7.73 (s, 1H), 7.45-7.49 (m, 2H), 7.33 (d, 2H, J=8.3 Hz), 7.25-7.30 (m, 1H), 3.93 (dd, 1H, J=7.2, 4.4 Hz), 3.07 (d, 1H, J=7.2 Hz), 2.50 (d, 1H, J=4.5 Hz), 2.42 (s, 3H) 
     Materials and Methods 
     The following paragraphs describe in more detail the materials and methods used in Examples 1-3. 
     General. 
     Unless otherwise noted, all chemicals and reagents for reactions were obtained from commercial suppliers (Sigma-Aldrich, VWR, Alfa Aesar) and used without further purification. Silica gel chromatography purifications were carried out using AMD Silica Gel 60, 230-400 mesh.  1 H spectra were recorded on a Varian Inova 300 MHz or Bruker Prodigy 400 MHz instrument in CDCl 3 , and are referenced to the residual solvent peak. Synthetic reactions were monitored using thin layer chromatography (Merck 60 gel plates) using an UV-lamp for visualization. 
     Chromatography. 
     Analytical high-performance liquid chromatography (HPLC) was carried out using an Agilent 1200 series, and a Kromasil 100 C18 column (4.6×50 mm, 5 μm). Semi-preparative HPLC was performed using an Agilent XDB-C18 (9.4×250 mm, 5 μm). Analytical chiral HPLC was conducted using a supercritical fluid chromatography (SFC) system with isopropanol and liquid CO 2  as the mobile phase. Chiral OB-H and AS-H columns were used to separate aziridine and amido-alcohol enantiomers (4.6×150 mm, 5 μm). Olefins were all commercially available; amido-alcohol and aziridine standards were prepared as reported. % ee was calculated by dividing the major peak area by the sum of the peak areas determined by SFC chromatography 
     Cloning and Site-Directed Mutagenesis. 
     pET22b(+) was used as a cloning and expression vector for all enzymes described in this study. Site-directed mutagenesis was performed using a modified QuikChange™ mutagenesis protocol. The PCR products were gel purified, digested with DpnI, repaired using Gibson Mix™, and directed transformed into  E. coli  strain BL21(DE3). 
     Determination of P450 Concentration. 
     Concentration of P450/P411 enzymes for in whole cell experiments was determined from ferrous carbon monoxide binding difference spectra using previously reported extinction coefficients for cysteine-ligated (c=91,000 M −1  cm −1 ) and serine-ligated enzymes (c=103,000 M −1  cm −1 ). When purified enzymes were used, concentration of P450/P411 enzymes was accomplished by quantifying the amount of free hemin present in purified protein using the pyridine/hemochrome assay. 
     Protein Expression and Purification. 
     Enzymes used in purified protein experiments were expressed in BL21(DE3)  E. coli  cultures transformed with plasmid encoding P450 or P411 variants. Expression and purification were performed as described except that the shake rate was lowered to 130 RPM during expression (Coelho, P. S., et al.  Science,  339, 307 (2013)). Following expression, cells were pelleted and frozen at −20 OC. For purification, frozen cells were resuspended in buffer A (20 mM tris, 20 mM imidazole, 100 mM NaCl, pH 7.5, 4 mL/g of cell wet weight), loaded with 300 μg/ml hemin, and disrupted by sonication (2×1 min, output control 5, 50% duty cycle; Sonicator 3000, Misonix, Inc.). To pellet insoluble material, lysates were centrifuged (20,000×g for 0.5 h at 4° C.). Proteins were expressed in a construct containing a 6×-His tag and were consequently purified using a nickel NTA column (1 mL HisTrap HP, GE Healthcare, Piscataway, N.J.) using an AKTAxpress purifier FPLC system (GE healthcare). P450 or P411 enzymes were then eluted on a linear gradient from 0% buffer B (20 mM tris, 300 mM imidazole, 100 mM NaCl, pH 7.5) to 100% buffer B over 10 column volumes (P450/P411 enzymes elute at around 80 mM imidazole). Fractions containing P450 or P411 enzymes were pooled, concentrated, and subjected to three exchanges of phosphate buffer (0.1 M KPi pH 8.0) to remove excess salt and imidazole. Concentrated proteins were aliquoted, flash-frozen on powdered dry ice, and stored at −20° C. until later use 
     Reaction Screening in 96-Well Plate Format. 
     Site-saturation mutagenesis libraries were generated by employing the “22c-trick” method (Kille, S., et al.,  ACS Synth. Biol.,  2, 83-92 (2013)).  E. coli  libraries were generated and cultured in 300 μL of LB with 100 ug/ml ampicillin and stored as glycerol stocks at −80° C. in 96-well plates. 50 μL of the pre-culture was transferred to a 1000 μL of Hyperbroth using a multichannel pipette. The cultures were incubated at 37° C., 220 rpm, 80% humidity for 3 hours. The plates were cooled on ice for 15 minutes before expression was induced (0.5 mM IPTG, 1 mM 5-aminolevulinic acid final concentration). Expression was conducted at 20° C., 120 rpm, 20 h. The cells were pelleted (3000×g, 5 min) and re-suspended in 40 μL/well GOX solution (14,000 U/ml catalase (Sigma 02071) and 1000 U/ml glucose oxidase (Sigma G7141)). The 96-well plate was transferred to an anaerobic chamber. To this mixture was added 300 μL per well argon sparged reaction buffer (4:1 M9-N: 250 mM glucose in M9-N) was added followed by 4-methylstyrene (300 mM, 10 μL/well) and tosyl azide (100 mM, 10 μL/well). The plate was sealed with aluminum sealing tape, removed from the anaerobic chamber, and shaken at 40 rpm. After 16 hours, the seal was removed and 400 μL of acetonitrile was added to each well. The contents of each well were mixed by pipetting up and down using a multichannel pipette. Then the plate was centrifuged (4000×g, 5 minutes) and 500 μL of the supernatant was transferred to a shallow-well plate for analysis by HPLC. 
     Typical Procedure for Small-Scale Aziridination Bioconversions Under Anaerobic Conditions Using Whole Cells and Purified Enzymes. 
       E. coli  BL21(DE3) cells containing P450 or P411 enzymes were grown from glycerol stock overnight (37° C., 250 rpm) in 5 ml Luria broth with 0.1 mg mL −1  ampicillin. The preculture was used to inoculate 45 mL of Hyperbroth medium (prepared from AthenaES© powder, 0.1 mg mL 1  ampicillin) in a 125 mL Erlenmeyer flask; this culture was incubated at 37° C., 220 rpm for 2 h and 30 min. After, the cultures were cooled on ice and induced with 0.5 mM IPTG and 1 mM 5-aminolevulinic acid (final concentration). Expression was conducted at room temperature, 120 rpm, 20 h. The cultures were then harvested and resuspended to OD 600 =30 in M9-N. Aliquots of the cell suspension (4 mL) were used for determination of the P450 or P411 expression level after lysis.  E. coli  cells (OD 600 =30) were made anaerobic by sparging with argon in a sealed 6 mL crimp vial for at least 30 minutes. To a 2 mL crimp vial was then added glucose (250 mM in M9-N, 40 μL) and the GOX solution described previously (20 μL). The headspace of the sealed 2 mL reaction vial was made anaerobic by flushing argon over the solution. Resuspended cells (320 μL), followed by olefin substrate (10 μL, 300 mM in DMSO), then tosyl azide (10 μL, 100 mM in DMSO) were added to 2 mL reaction vial via syringe under continuous flow of argon. Final concentrations of reagents were typically: 2.5 mM tosyl azide, 7.5 mM olefin, 25 mM glucose. The no enzyme control experiment was conducted using  E. coli  BL21 (DE3) cells containing empty pET22b(+) vector with the same reaction conditions as described above. Purified enzyme reactions were conducted as described previously, using 2.5 mM TsN 3  and 7.5 mM olefin (Farwell, C. C. et al.  J. Am. Chem. Soc.,  136, 8766-8771 (2014)). Sodium dithionite (5 mM) was used as reductant for reactions with hemin, myoglobin, cytochrome C, CYP119, and P450 Rhf . The reactions were shaken on a table-top shake plate (40 rpm) at room temperature for 4 hours. The reactions were quenched by adding acetonitrile (460 μL) and the resulting mixture was transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for 5 minutes. The solution (540 μL) was transferred to an HPLC vial, charged with internal standard (60 μL, 10 mM 1,3,5-trichlorobenzene in acetonitrile), and analyzed by HPLC. 
     Reactions for chiral HPLC analysis were performed on a 2 mL scale with the same concentration of reagents and using a similar procedure as described above. Briefly, cells containing P450 or P411 enzymes were expressed and resuspended to an OD 600 =30 in M9-N, and then degassed by sparging with argon in a sealed 6 mL crimp vial for at least 30 minutes. To a 6 mL crimp vial was then added glucose (250 mM in M9-N, 200 μL) and the GOX mixture described previously (100 μL). The headspace of the sealed 2 mL reaction vial was made anaerobic by flushing argon over the solution. Resuspended cells (1600 μL), followed by olefin substrate (50 μL, 300 mM in DMSO), then tosyl azide (50 μL, 100 mM in DMSO) were added to 6 mL reaction vial via syringe under continuous flow of argon. Reactions were quenched with 2 mL acetonitrile, extracted with ethyl acetate, dried and resuspended in acetone (200 μL), and purified by C18 semi-preparative HPLC. The purified material was dried, resuspended in acetonitrile, and analyzed by SFC for enantioselectivity. 
     Determination of Initial Rates. 
     All initial rate experiments were conducted in an anaerobic chamber. Initial rate measurements were accomplished using 0.2 mol % purified enzymes in 400 μL scale reactions. A sealed 6-mL vial charged with glucose (250 mM, 480 μL), NADPH (100 mM, 480 μL), and potassium phosphate buffer (0.1 M, pH=8.0, 3240 μL) was sparged for at least 30 minutes with argon. After the degassing was complete, the reaction solution, 2-mL vials charged with GOX solution (20 μL), and purified protein (250 μM in potassium phosphate buffer), kept on ice, were brought into the anaerobic chamber. The reaction solution (350 μL) was added to each 2-mL vial and allowed to equilibrate in the anaerobic chamber for 30 minutes. Reaction vials were then placed on a shaker (40 rpm), charged with 10 μL purified protein (250 μM in potassium phosphate buffer) and 4-methyl styrene substrate (10 μL, 300 mM in DMSO) followed by tosyl azide (10 μL, 100 mM in DMSO). Reactions were set up in duplicate and products quantified at 1-2 minute intervals by quenching with acetonitrile (460 μL). The resulting mixture was removed from the anaerobic chamber, transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for 5 minutes. The solution (540 μL) was transferred to an HPLC vial, charged with internal standard (60 μL, 10 mM 1,3,5-trichlorobenzene in acetonitrile), and analyzed by HPLC. The rates of aziridination and azide reduction for different enzyme variants are presented in  FIG. 6 . The rate of azide reduction was determined in the presence of olefin 3 (7.5 mM). Initial rates are plotted for individual enzymes in  FIG. 7  A-C. 
     Assignment of Absolute Stereochemistry. 
     Absolute stereochemistry of enzymatically produced aziridine 6 was assigned by chiral HPLC analysis and optical rotation. In particular, absolute stereochemistry of 6 was previously assigned by chiral HPLC using Chiracel OJ column (isopropanol/n-hexane mobile phase), with (S)-6 the earlier eluting enantiomer (Takeda Y.,  J. Am. Chem. Soc.,  136, 8544-7 (2014)). Analytically enantiopure 6 produced by P-I263F-A328V-L437V was subjected to the same chiral HPLC conditions and observed to be the earlier eluting enantiomer ( FIGS. 9A-B ), leading to an assignment of (S)-6. Further support for this assignment came from measuring optical rotation. The optical rotation values for enantiomers of 7 have been previously reported (R)-6 [α D   25 ]−80.25 (c=0.8, CHCl 3 ) and (S)-6 [α D   20 ]+26.7 (c=0.7, CHCl 3 ) (Alonso, D. A., et al.,  J. Org. Chem.,  63, 9455-9461 (1998); Wang, X. et al.,  Chem. Eur. J.,  12, 4568-4575 (2006)). Optical rotation measurement of analytically enantiopure 6 produced by P-I263F-A328V-L437V gave [α D   25 ]+80.2 (c=1.2, CHCl 3 ), revealing it to be (S)-6. Similarly, the optical rotation of P-I263F-A328V-L437V produced 4 (analytically enantiopure) was measured to be [α D   25 ]+106.1 (c=0.45, CHCl 3 ). By analogy, the configuration of enzymatically preferred (+)-4 is assigned as (S)-4. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may 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. 
     Informal Sequence Listing 
       
     
       
         
           
               
               
            
               
                 CYP102A1 
                   
               
               
                 Cytochrome P450 (BM3) 
               
               
                 
                   Bacillus megaterium 
                 
               
               
                 GenBank Accession No. AAA87602 
               
               
                 &gt;gi|142798|gb|AAA87602.1| cytochrome P-450:NADPH-P-450 reductase  
               
               
                 precursor [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 1 
                   
               
               
                  TIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 
                   
               
               
                   
               
               
                 EACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 
               
               
                   
               
               
                 VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 
               
               
                   
               
               
                 ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 
               
               
                   
               
               
                 GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 
               
               
                   
               
               
                 DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 
               
               
                   
               
               
                 LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 
               
               
                   
               
               
                 HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 
               
               
                   
               
               
                 TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 
               
               
                   
               
               
                 PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 
               
               
                   
               
               
                 RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 
               
               
                   
               
               
                 GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 
               
               
                   
               
               
                 LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 
               
               
                   
               
               
                 LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 
               
               
                   
               
               
                 MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 
               
               
                   
               
               
                 LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 
               
               
                   
               
               
                 VHQVSEADAR LWLQQLEEKG RYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191140|gb|ADA57069.1| NADPH-cytochrome P450 reductase 102A1V9 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 2 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN 
               
               
                   
               
               
                 LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191138|gb|ADA57068.1| NADPH-cytochrome P450 reductase 102A1V10 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 3 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKFVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAFEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVFGCGDKNWATTYQKVPAFIDETFAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN 
               
               
                   
               
               
                 LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVATRFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191126|gb|ADA57062.1| NADPH-cytochrome P450 reductase 102A1V4 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 4 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYEN 
               
               
                   
               
               
                 LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSERSTRHLEIALPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVTAREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSIRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRSFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191124|gb|ADA57061.1| NADPH-cytochrome P450 reductase 102A1V8 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 5 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPRVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN 
               
               
                   
               
               
                 LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191120|gb|ADA57059.1| NADPH-cytochrome P450 reductase 102A1V3 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 6 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYEN 
               
               
                   
               
               
                 LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVTAREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 ERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191118|gb|ADA57058.1| NADPH-cytochrome P450 reductase 102A1V7 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 7 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPPEGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN 
               
               
                   
               
               
                 LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNEPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191112|gb|ADA57055.1| NADPH-cytochrome P450 reductase 102A1V2 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 8 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEATRVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGEDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADDV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYEN 
               
               
                   
               
               
                 LDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQLGSERSTRHLEIALPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVTARFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSISPRYYSISSSPHVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKDSETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 ERDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYADVYEVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|269315992|gb|ACZ37122.1| cytochrome P450:NADPH P450 reductase  
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 9 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPIQTLMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNTDEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKEFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLAAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN 
               
               
                   
               
               
                 LDIENSEENASTLSLQFVDSAADMPLAKMHRAFSANVVASKELQKPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVATREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEVLLEKQAYKEQVLAKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLANLQEGDTITCFVSTPQSGFTLPKGPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQKELENAQNEGIITLHTAFSRVPNQPKTYVQHVM 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHQVSEADARLWLQQLEEKGRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191116|gb|ADA57057.1| NADPH-cytochrome P450 reductase 102A1V6 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 10 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HEDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVEGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYEN 
               
               
                   
               
               
                 LNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVTTREGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVV 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAG 
               
               
                   
               
               
                 CYP102A1 
               
               
                 
                   B. megaterium 
                 
               
               
                 &gt;gi|281191114|gb|ADA57056.1| NADPH-cytochrome P450 reductase 102A1V5 
               
               
                 [ Bacillus megaterium ] 
               
               
                 SEQ ID NO: 11 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDK 
                   
               
               
                   
               
               
                 NLSQALKEVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLIQKWERLNADEHI 
               
               
                   
               
               
                 EVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQDDI 
               
               
                   
               
               
                 KVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYF 
               
               
                   
               
               
                 LVKNPHVLQKAAFEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPLEK 
               
               
                   
               
               
                 GDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLK 
               
               
                   
               
               
                 HFDFEDHTNYELDIKETLTLKPEGFVVKAKSKQIPLGGIPSPSREQSAKKERKTVENAHNTPLLVLYGSN 
               
               
                   
               
               
                 MGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEV 
               
               
                   
               
               
                 KGVRYSVFGCGDKNWATTYQKVPAFIDETLSAKGAENIAERGEADASDDFEGTYEEWREHMWSDLAAYFN 
               
               
                   
               
               
                 LNIENSEDNASTLSLQFVDSAADMPLAKMHGAFSANVVASKELQQPGSARSTRHLEIELPKEASYQEGDH 
               
               
                   
               
               
                 LGVIPRNYEGIVNRVTTRFGLDASQQIRLEAFEEKLAHLPLGKTVSVEELLQYVELQDPVTRTQLRAMAA 
               
               
                   
               
               
                 KTVCPPHKVELEALLEKQAYKEQVLTKRLTMLELLEKYPACEMEFSEFIALLPSMRPRYYSISSSPRVDE 
               
               
                   
               
               
                 KQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFVSTPQSGFTLPKDPETPLIMVGPGTGVAP 
               
               
                   
               
               
                 FRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQNEGIITLHTAFSRVPNQPKTYVQHVV 
               
               
                   
               
               
                 EQDGKKLIELLDQGAHFYICGDGSQMAPDVEATLMKSYAEVHKVSEADARLWLQQLEEKSRYAKDVWAG 
               
               
                   
               
               
                 CYP153A6 
               
               
                   Mycobacterium  sp. HXN-1500 
               
               
                 GenBank Accession No.: CAH04396 
               
               
                 &gt;gi|51997117|emb|CAH04396.1| cytochrome P450 alkane hydroxylase 
               
               
                 [ Mycobacterium  sp. HXN-1500] 
               
               
                 SEQ ID NO: 12 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MTEMTVAASD ATNAAYGMAL EDIDVSNPVL FRDNTWHPYF KRLREEDPVH YCKSSMFGPY 
                   
               
               
                   
               
               
                  61 
                 WSVTKYRDIM AVETNPKVFS SEAKSGGITI MDDNAAASLP MFIAMDPPKH DVQRKTVSPI 
               
               
                   
               
               
                 121 
                 VAPENLATME SVIRQRTADL LDGLPINEEF DWVHRVSIEL TTKMLATLFD FPWDDRAKLT 
               
               
                   
               
               
                 181 
                 RWSDVTTALP GGGIIDSEEQ RMAELMECAT YFTELWNQRV NAEPKNDLIS MMAHSESTRH 
               
               
                   
               
               
                 241 
                 MAPEEYLGNI VLLIVGGNDT TRNSMTGGVL ALNEFPDEYR KLSANPALIS SMVSEIIRWQ 
               
               
                   
               
               
                 301 
                 TPLSHMRRTA LEDIEFGGKH IRQGDKVVMW YVSGNRDPEA IDNPDTFIID RAKPRQHLSF 
               
               
                   
               
               
                 361 
                 GFGIHRCVGN RLAELQLNIL WEEILKRWPD PLQIQVLQEP TRVLSPFVKG YESLPVRINA 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP5013C2 
                   
               
               
                 
                   Tetrahymena thermophile 
                 
               
               
                 GenBank Accession No.: ABY59989 
               
               
                 &gt;gi|164519863|gb|ABY59989.1| cytochrome P450 monooxygenase CYP5013C2 
               
               
                   [Tetrahymena thermophila ] 
               
               
                 SEQ ID NO: 13 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MIFELILIAV ALFAYFKIAK PYFSYLKYRK YGKGFYYPIL GEMIEQEQDL KQHADADYSV 
                   
               
               
                   
               
               
                  61 
                 HHALDKDPDQ KLFVTNLGTK VKLRLIEPEI IKDFFSKSQY YQKDQTFIQN ITRFLKNGIV 
               
               
                   
               
               
                 121 
                 FSEGNTWKES RKLFSPAFHY EYIQKLTPLI NDITDTIFNL AVKNQELKNF DPIAQIQEIT 
               
               
                   
               
               
                 181 
                 GRVIIASFFG EVIEGEKFQG LTIIQCLSHI INTLGNQTYS IMYFLFGSKY FELGVTEEHR 
               
               
                   
               
               
                 241 
                 KFNKFIAEFN KYLLQKIDQQ IEIMSNELQT KGYIQNPCIL AQLISTHKID EITRNQLFQD 
               
               
                   
               
               
                 301 
                 FKTFYIAGMD TTGHLLGMTI YYVSQNKDIY TKLQSEIDSN TDQSAHGLIK NLPYLNAVIK 
               
               
                   
               
               
                 361 
                 ETLRYYGPGN ILFDRIAIKD HELAGIPIKK GTIVTPYAMS MQRNSKYYQD PHKYNPSRWL 
               
               
                   
               
               
                 421 
                 EKQSSDLHPD ANIPFSAGQR KCIGEQLALL EARIILNKFI KMFDFTCPQD YKLMMNYKFL 
               
               
                   
               
               
                 481 
                 SEPVNPLPLQ LTLRKQ 
               
               
                   
               
            
           
           
               
               
            
               
                 
                   Nonomuraea dietziae 
                 
                   
               
               
                 &gt;gi|445067389|gb|AGE14547.1| cytochrome P450 hydroxylase sb8  
               
               
                 [ Nonomuraea dietziae ] 
               
               
                 GenBank Accession No.: AGE14547 
               
               
                 SEQ ID NO: 14 
                   
               
               
                 VNIDLVDQDHYATEGPPHEQMRWLREHAPVYWHEGEPGFWAVTRHEDVVHVSRHSDLESSARRLALFNEMPEEQREL 
                   
               
               
                   
               
               
                 QRMMMLNQDPPEHTRRRSLVNRGFTPRTIRALEQHIRDICDDLLDQCSGEGDFVTDLAAPLPLYVICELLGAPVADR 
               
               
                   
               
               
                 DKIFAWSNRMIGAQDPDYAASPEEGGAAAMEVYAYASELAAQRRAAPRDDIVTKLLQSDENGESLTENEFELFVLLL 
               
               
                   
               
               
                 VVAGNETTRNAASGGMLTLFEHPDQWDRLVADPSLAATAADEIVRWVSPVNLFRRTATADLTLGGQQVKADDKVVVF 
               
               
                   
               
               
                 YSSANRDASVESDPEVEDIGRSPNPHIGEGGGGAHFCLGNHLAKLELRVLFEQLARREPRMRQTGEARRLRSNFING 
               
               
                   
               
               
                 IKTLPVTLG 
               
               
                   
               
               
                 CYP2R1 
               
               
                 
                   Homo sapiens 
                 
               
               
                 GenBank Accession No.: NP 078790 
               
               
                 &gt;gi|45267826|ref|NP_078790.2| vitamin D 25-hydroxylase [ Homo sapiens ] 
               
               
                 SEQ ID NO: 15 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MWKLWRAEEG AAALGGALFL LLFALGVRQL LKQRRPMGFP PGPPGLPFIG NIYSLAASSE 
                   
               
               
                   
               
               
                  61 
                 LPHVYMRKQS QVYGEIFSLD LGGISTVVLN GYDVVKECLV HQSEIFADRP CLPLFMKMTK 
               
               
                   
               
               
                 121 
                 MGGLLNSRYG RGWVDHRRLA VNSFRYFGYG QKSFESKILE ETKFFNDAIE TYKGRPFDFK 
               
               
                   
               
               
                 181 
                 QLITNAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGILPF 
               
               
                   
               
               
                 241 
                 GKHQQLFRNA AVVYDFLSRL IEKASVNRKP QLPQHFVDAY LDEMDQGKND PSSTFSKENL 
               
               
                   
               
               
                 301 
                 IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPNGKP SWDDKCKMPY 
               
               
                   
               
               
                 361 
                 TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRDPEVF 
               
               
                   
               
               
                 421 
                 HPERFLDSSG YFAKKEALVP FSLGRRHCLG EHLARMEMFL FFTALLQRFH LHFPHELVPD 
               
               
                   
               
               
                 481 
                 LKPRLGMTLQ PQPYLICAER R 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP2R1 
                   
               
               
                 
                   Macca mulatta 
                 
               
               
                 GenBank Accession No.: NP 001180887 
               
               
                 &gt;gi|302565346|ref|NP_001180887.1| vitamin D 25-hydroxylase  
               
               
                 [ Macaca mulatta ] 
               
               
                 SEQ ID NO: 16 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MWKLWGGEEG AAALGGALFL LLFALGVRQL LKLRRPMGFP PGPPGLPFIG NIYSLAASAE 
                   
               
               
                   
               
               
                  61 
                 LPHVYMRKQS QVYGEIFSLD LGGISTVVLN GYDVVKECLV HQSGIFADRP CLPLFMKMTK 
               
               
                   
               
               
                 121 
                 MGGLLNSRYG QGWVEHRRLA VNSFRYFGYG QKSFESKILE ETKFFTDAIE TYKGRPFDFK 
               
               
                   
               
               
                 181 
                 QLITSAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGILPF 
               
               
                   
               
               
                 241 
                 GKHQQLFRNA SVVYDFLSRL IEKASVNRKP QLPQHFVDAY FDEMDQGKND PSSTFSKENL 
               
               
                   
               
               
                 301 
                 IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPNGKP SWDDKFKMPY 
               
               
                   
               
               
                 361 
                 TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRDPEVF 
               
               
                   
               
               
                 421 
                 HPERFLDSSG YFAKKEALVP FSLGRRHCLG EQLARMEMFL FFTALLQRFH LHFPHELVPD 
               
               
                   
               
               
                 481 
                 LKPRLGMTLQ PQPYLICAER R 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP2R1 
                   
               
               
                 
                   Canis familiaris 
                 
               
               
                 GenBank Accession No.: XP_854533 
               
               
                 &gt;gi|73988871|ref|XP_854533.11 PREDICTED: vitamin D 25-hydroxylase  
               
               
                 [ Canis   lupus familiaris ] 
               
               
                 SEQ ID NO: 17 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MRGPPGAEAC AAGLGAALLL LLFVLGVRQL LKQRRPAGFP PGPSGLPFIG NIYSLAASGE 
                   
               
               
                   
               
               
                  61 
                 LAHVYMRKQS RVYGEIFSLD LGGISAVVLN GYDVVKECLV HQSEIFADRP CLPLFMKMTK 
               
               
                   
               
               
                 121 
                 MGGLLNSRYG RGWVDHRKLA VNSFRCFGYG QKSFESKILE ETNFFIDAIE TYKGRPFDLK 
               
               
                   
               
               
                 181 
                 QLITNAVSNI TNLIIFGERF TYEDTDFQHM IELFSENVEL AASASVFLYN AFPWIGIIPF 
               
               
                   
               
               
                 241 
                 GKHQQLFRNA AVVYDFLSRL IEKASINRKP QSPQHFVDAY LNEMDQGKND PSCTFSKENL 
               
               
                   
               
               
                 301 
                 IFSVGELIIA GTETTTNVLR WAILFMALYP NIQGQVQKEI DLIMGPTGKP SWDDKCKMPY 
               
               
                   
               
               
                 361 
                 TEAVLHEVLR FCNIVPLGIF HATSEDAVVR GYSIPKGTTV ITNLYSVHFD EKYWRNPEIF 
               
               
                   
               
               
                 421 
                 YPERFLDSSG YFAKKEALVP FSLGKRHCLG EQLARMEMFL FFTALLQRFH LHFPHGLVPD 
               
               
                   
               
               
                 481 
                 LKPRLGMTLQ PQPYLICAER R 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP2R1 
                   
               
               
                 
                   Mus musculus 
                 
               
               
                 GenBank Accession No.: AAI08963 
               
               
                 &gt;gi|80477959|gb|AAI08963.1| Cyp2r1 protein [ Mus musculus ] 
               
               
                 SEQ ID NO: 18 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MGDEMDQGQN DPLSTFSKEN LIFSVGELII AGTETTTNVL RWAILFMALY PNIQGQVHKE 
                   
               
               
                   
               
               
                  61 
                 IDLIVGHNRR PSWEYKCKMP YTEAVLHEVL RFCNIVPLGI FHATSEDAVV RGYSIPKGTT 
               
               
                   
               
               
                 121 
                 VITNLYSVHF DEKYWKDPDM FYPERFLDSN GYFTKKEALI PFSLGRRHCL GEQLARMEMF 
               
               
                   
               
               
                 181 
                 LFFTSLLQQF HLHFPHELVP NLKPRLGMTL QPQPYLICAE RR 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP152A6 
                   
               
               
                   Bacillus halodurans  C-125 
               
               
                 GenBank Accession No.: NP|242623 
               
               
                 &gt;gi|15614320|ref|NP|242623.11 fatty acid alpha hydroxylase  
               
               
                 [ Bacillus   halodurans  C-125] 
               
               
                 SEQ ID NO: 19 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MKSNDPIPKD SPLDHTMNLM REGYEFLSHR MERFQTDLFE TRVMGQKVLC IRGAEAVKLF 
                   
               
               
                   
               
               
                  61 
                 YDPERFKRHR ATPKRIQKSL FGENAIQTMD DKAHLHRKQL FLSMMKPEDE QELARLTHET 
               
               
                   
               
               
                 121 
                 WRRVAEGWKK SRPIVLFDEA KRVLCQVACE WAEVPLKSTE IDRRAEDFHA MVDAFGAVGP 
               
               
                   
               
               
                 181 
                 RHWRGRKGRR RTERWIQSII HQVRTGSLQA REGSPLYKVS YHRELNGKLL DERMAAIELI 
               
               
                   
               
               
                 241 
                 NVLRPIVAIA TFISFAAIAL QEHPEWQERL KNGSNEEFHM FVQEVRRYYP FAPLIGAKVR 
               
               
                   
               
               
                 301 
                 KSFTWKGVRF KKGRLVFLDM YGTNHDPKLW DEPDAFRPER FQERKDSLYD FIPQGGGDPT 
               
               
                   
               
               
                 361 
                 KGHRCPGEGI TVEVMKTTMD FLVNDIDYDV PDQDISYSLS RMPTRPESGY IMANIERKYE 
               
               
                   
               
               
                 421 
                 HA 
               
               
                   
               
            
           
           
               
               
            
               
                 aryC 
                   
               
               
                 
                   Streptomyces parvus 
                 
               
               
                 GenBank Accession No.: AFM80022 
               
               
                 &gt;gi|392601346|gb|AFM80022.1| cytochrome P450 +Streptomyces parvus+ 
               
               
                 SEQ ID NO: 20 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MYLGGRRGTE AVGESREPGV WEVFRYDEAV QVLGDHRTFS SDMNHFIPEE QRQLARAARG 
                   
               
               
                   
               
               
                  61 
                 NFVGIDPPDH TQLRGLVSQA FSPRVTAALE PRIGRLAEQL LDDIVAERGD KASCDLVGEF 
               
               
                   
               
               
                 121 
                 AGPLSAIVIA ELFGIPESDH TMIAEWAKAL LGSRPAGELS IADEAAMQNT ADLVRRAGEY 
               
               
                   
               
               
                 181 
                 LVHHITERRA RPQDDLTSRL ATTEVDGKRL DDEEIVGVIG MFLIAGYLPA SVLTANTVMA 
               
               
                   
               
               
                 241 
                 LDEHPAALAE VRSDPALLPG AIEEVLRWRP PLVRDQRLTT RDADLGGRTV PAGSMVCVWL 
               
               
                   
               
               
                 301 
                 ASAHRDPFRF ENPDLFDIHR NAGRHLAFGK GIHYCLGAPL ARLEARIAVE TLLRRFERIE 
               
               
                   
               
               
                 361 
                 IPRDESVEFH ESIGVLGPVR LPTTLFARR 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP101A1 
                   
               
               
                 
                   Pseudomonas putida 
                 
               
               
                 Uniprot Accession No.: P00183 
               
               
                 &gt;sp|P00183|CPXA_PSEPU Camphor 5-monooxygenase OS =  Pseudomonas putida   
               
               
                 GN = camC PE = 1 SV = 2 
               
               
                 SEQ ID NO: 21 
                   
               
               
                 TTETIQSNANLAPLPPHVPEHLVEDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYED 
                   
               
               
                   
               
               
                 YRHESSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDY 
               
               
                   
               
               
                 AEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGR 
               
               
                   
               
               
                 PITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSLVADGRILTSDYEFH 
               
               
                   
               
               
                 GVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAP 
               
               
                   
               
               
                 GAQIQHKSGIVSGVQALPLVWDPATTKAV 
               
               
                   
               
               
                 
                   Homo sapiens 
                 
               
               
                 CYP2D7 
               
               
                 GenBank Accession No.: AA049806 
               
               
                 &gt;gi|37901459|gb|AA049806.1| cytochrome P450 [ Homo sapiens ] 
               
               
                 SEQ ID NO: 22 
                   
               
               
                 GLEALVPLA MIVAIFLLLV DLMHRHQRWA ARYPPGPLPL PGLGNLLHVD FQNTPYCFDQ 
                   
               
               
                   
               
               
                 LRRRFGDVFN LQLAWTPVVV LNGLAAVREA MVTRGEDTAD RPPAPIYQVL GFGPRSQGVI 
               
               
                   
               
               
                 LSRYGPAWRE QRRFSVSTLR NLGLGKKSLE QWVTEEAACL CAAFADQAGR PFRPNGLLDK 
               
               
                   
               
               
                 AVSNVIASLT CGRRFEYDDP RFLRLLDLAQ EGLKEESGFL REVLNAVPVL PHIPALAGKV 
               
               
                   
               
               
                 LRFQKAFLTQ LDELLTEHRM TWDPAQPPRD LTEAFLAKKE KAKGSPESSF NDENLRIVVG 
               
               
                   
               
               
                 NLFLAGMVTT LTTLAWGLLL MILHLDVQRG RRVSPGCSPI VGTHVCPVRV QQEIDDVIGQ 
               
               
                   
               
               
                 VRRPEMGDQV HMPYTTAVIH EVQRFGDIVP LGVTHMTSRD IEVQGFRIPK GTTLITNLSS 
               
               
                   
               
               
                 VLKDEAVWEK PFRFHPEHFL DAQGHFVKPE AFLPFSAGRR ACLGEPLARM ELFLFFTSLL 
               
               
                   
               
               
                 QHFSFSVAAG QPRPSHSRVV SFLVTPSPYE LCAVPR 
               
               
                   
               
               
                 
                   Rattus norvegicus 
                 
               
               
                 CYPC27 
               
               
                 GenBank Accession No.: AAB02287 
               
               
                 &gt;gi|1374714|gb|AAB02287.1| cytochrome P450 [ Rattus norvegicus ] 
               
               
                 SEQ ID NO: 23 
                   
               
               
                 AVLSRMRLRWALLDTRVMGHGLCPQGARAKAAIPAALRDHESTEGPGTGQDRPRLRSLAELPGPGTLRF 
                   
               
               
                   
               
               
                 LFQLFLRGYVLHLHELQALNKAKYGPMWTTTEGTRTNVNLASAPLLEQVMRQEGKYPIRDSMEQWKEHRD 
               
               
                   
               
               
                 HKGLSYGIFITQGQQWYHLRHSLNQRMLKPAEAALYTDALNEVISDFIARLDQVRTESASGDQVPDVAHL 
               
               
                   
               
               
                 LYHLALEAICYILFEKRVGCLEPSIPEDTATFIRSVGLMEKNSVYVTFLPKWSRPLLPFWKRYMNNWDNI 
               
               
                   
               
               
                 FSFGEKMIHQKVQEIEAQLQAAGPDGVQVSGYLHFLLTKELLSPQETVGTFPELILAGVDTTSNTLTWAL 
               
               
                   
               
               
                 YHLSKNPEIQEALHKEVTGVVPFGKVPQNKDFAHMPLLKAVIKETLRLYPVVPTNSRIITEKETEINGFL 
               
               
                   
               
               
                 FPKNTQFVLCTYVVSRDPSVFPEPESFQPHRWLRKREDDNSGIQHPFGSVPFGYGVRSCLGRRIAELEMQ 
               
               
                   
               
               
                 LLLSRLIQKYEVVLSPGMGEVKSVSRIVLVPSKKVSLRFLQRQ 
               
               
                   
               
               
                 CYP2B4 
               
               
                 
                   Oryctolagus cuniculus 
                 
               
               
                 GenBank Accession No. AAA65840 
               
               
                 &gt;gi|164959|gb|AAA65840.1| cytochrome P-450 [ Oryctolagus cuniculus ] 
               
               
                 SEQ ID NO: 24 
                   
               
               
                 MEFSLLLLLAFLAGLLLLLFRGHPKAHGRLPPGPSPLPVLGNLLQMDRKGLLRSFLRLRE 
                   
               
               
                   
               
               
                 KYGDVFTVYLGSRPVVVLCGTDAIREALVDQAEAFSGRGKIAVVDPIFQGYGVIFANGER 
               
               
                   
               
               
                 WRALRRFSLATMRDFGMGKRSVEERIQEEARCLVEELRKSKGALLDNTLLFHSITSNIIC 
               
               
                   
               
               
                 SIVEGKREDYKDPVFLRLLDLFFQSFSLISSFSSQVFELFPGFLKHFPGTHRQIYRNLQE 
               
               
                   
               
               
                 INTFIGQSVEKHRATLDPSNPRDFIDVYLLRMEKDKSDPSSEFHHQNLILTVLSLFFAGT 
               
               
                   
               
               
                 ETTSTTLRYGELLMLKYPHVTERVQKEIEQVIGSHRPPALDDRAKMPYTDAVIHEIQRLG 
               
               
                   
               
               
                 DLIPFGVPHTVTKDTQFRGYVIPKNTEVFPVLSSALHDPRYFETPNTENPGHFLDANGAL 
               
               
                   
               
               
                 KRNEGFMPFSLGKRICLGEGIARTELFLFFTTILQNFSIASPVPPEDIDLTPRESGVGNV 
               
               
                   
               
               
                 PPSYQIRFLAR 
               
               
                   
               
               
                 CYP102A2 
               
               
                 
                   Bacillus subtilis 
                 
               
               
                 Uniprot Accession No. 008394 
               
               
                 &gt;sp|O08394|CYPD_BACSU Probable bifunctional P-450/NADPH-P450  
               
               
                 reductase 1 OS =  Bacillus subtilis  (strain 168) GN = cypD  
               
               
                 PE = 3 SV = 1 
               
               
                 SEQ ID NO: 25 
                   
               
               
                 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELV 
                   
               
               
                   
               
               
                 KEVCDEERFDKSIEGALEKVRAFSGDGLFTSWTHEPNWRKAHNILMPTESQRAMKDYHEK 
               
               
                   
               
               
                 MVDIAVQLIQKWARLNPNEAVDVPGDMTRLTLDTIGLCGFNYRFNSYYRETPHPFINSMV 
               
               
                   
               
               
                 RALDEAMHQMQRLDVQDKLMVRTKRQFRHDIQTMESLVDSIIAERRANGDQDEKDLLARM 
               
               
                   
               
               
                 LNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRV 
               
               
                   
               
               
                 LTDAAPTYKQVLELTYIRMILNESLRLWPTAPAFSLYPKEDTVIGGKFPITTNDRISVLI 
               
               
                   
               
               
                 PQLHRDRDAWGKDAEEFRPERFEHQDQVPHHAYKPFGNGQRACIGMQFALHEATLVLGMI 
               
               
                   
               
               
                 LKYFTLIDHENYELDIKQTLTLKPGDFHIRVQSRNQDAIHADVQAVEKAASDEQKEKTEA 
               
               
                   
               
               
                 KGTSVIGLNNRPLLVLYGSDTGTAEGVARELADTASLHGVRTETAPLNDRIGKLPKEGAV 
               
               
                   
               
               
                 VIVTSSYNGKPPSNAGQFVQWLQEIKPGELEGVHYAVEGCGDHNWASTYQYVPRFIDEQL 
               
               
                   
               
               
                 AEKGATRFSARGEGDVSGDFEGQLDEWKKSMWADAIKAFGLELNENADKERSTLSLQFVR 
               
               
                   
               
               
                 GLGESPLARSYEASHASIAENRELQSADSDRSTRHIEIALPPDVEYQEGDHLGVLPKNSQ 
               
               
                   
               
               
                 TNVSRILHREGLKGTDQVTLSASGRSAGHLPLGRPVSLHDLLSYSVEVQEAATRAQIREL 
               
               
                   
               
               
                 AAFTVCPPHRRELEELSAEGVYQEQILKKRISMLDLLEKYEACDMPFERFLELLRPLKPR 
               
               
                   
               
               
                 YYSISSSPRVNPRQASITVGVVRGPAWSGRGEYRGVASNDLAERQAGDDVVMFIRTPESR 
               
               
                   
               
               
                 FQLPKDPETPIIMVGPGTGVAPERGELQARDVLKREGKTLGEAHLYFGCRNDRDFIYRDE 
               
               
                   
               
               
                 LERFEKDGIVTVHTAFSRKEGMPKTYVQHLMADQADTLISILDRGGRLYVCGDGSKMAPD 
               
               
                   
               
               
                 VEAALQKAYQAVHGTGEQEAQNWLRHLQDTGMYAKDVWAGI 
               
               
                   
               
               
                 CYP102A3 
               
               
                 
                   Bacillus subtilis 
                 
               
               
                 Uniprot Accession No.008336 
               
               
                 &gt;sp|008336|CYPE_BACSU Probable bifunctional P-450/NADPH-P450  
               
               
                 reductase 2 OS =  Bacillus subtilis  (strain 168) GN = cypE  
               
               
                 PE = 2 SV = 1 
               
               
                 SEQ ID NO: 26 
                   
               
               
                 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLV 
                   
               
               
                   
               
               
                 AEVCDESREDKNLGKGLQKVREFGGDGLFTSWTHEPNWQKAHRILLPSFSQKAMKGYHSM 
               
               
                   
               
               
                 MLDIATQLIQKWSRLNPNEEIDVADDMTRLTLDTIGLCGFNYRFNSFYRDSQHPFITSML 
               
               
                   
               
               
                 RALKEAMNQSKRLGLQDKMMVKTKLQFQKDIEVMNSLVDRMIAERKANPDDNIKDLLSLM 
               
               
                   
               
               
                 LYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRV 
               
               
                   
               
               
                 LTDDTPEYKQIQQLKYTRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLI 
               
               
                   
               
               
                 PKLHRDQNAWGPDAEDFRPERFEDPSSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLV 
               
               
                   
               
               
                 LKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQRKEQADIKAETKPKETKP 
               
               
                   
               
               
                 KHGTPLLVLYGSNLGTAEGIAGELAAQGRQMGETAETAPLDDYIGKLPEEGAVVIVTASY 
               
               
                   
               
               
                 NGSPPDNAAGFVEWLKELEEGQLKGVSYAVEGCGNRSWASTYQRIPRLIDDMMKAKGASR 
               
               
                   
               
               
                 LTE IGEGDAADDFESHRESWENRFWKETMDAFDINEIAQKEDRPSLSIAFLSEATETPVA 
               
               
                   
               
               
                 KAYGAFEGVVLENRELQTADSTRSTRHIELEIPAGKTYKEGDHIGIMPKNSRELVQRVLS 
               
               
                   
               
               
                 REGLQSNHVIKVSGSAHMSHLPMDRPIKVADLLSSYVELQEPASRLQLRELASYTVCPPH 
               
               
                   
               
               
                 QKELEQLVLDDGIYKEQVLAKRLTMLDFLEDYPACEMPFERFLALLPSLKPRYYSISSSP 
               
               
                   
               
               
                 KVHANIVSMTVGVVKASAWSGRGEYRGVASNYLAELNTGDAAACFIRTPQSGFQMPDEPE 
               
               
                   
               
               
                 TPMIMVGPGTGIAPERGFIQARSVLKKEGSTLGEALLYFGCRRPDHDDLYREELDQAEQE 
               
               
                   
               
               
                 GLVTIRRCYSRVENESKGYVQHLLKQDSQKLMTLIEKGAHIYVCGDGSQMAPDVEKTLRW 
               
               
                   
               
               
                 AYETEKGASQEESADWLQKLQDQKRYIKDVWTGN 
               
               
                   
               
               
                 CYP102A1 
               
               
                   B. megaterium  DSM 32 
               
               
                 Uniprot Accession No. P14779 
               
               
                 &gt;sp|P14779|CPXB_BACME Bifunctional P-450/NADPH-P450 reductase OS+32 Bacillus   
               
               
                   megaterium  GN = cyp102A1 PE = 1 SV = 2 
               
               
                 SEQ ID NO: 27 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK 
                   
               
               
                   
               
               
                   61 
                 EACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM 
               
               
                   
               
               
                  121 
                 VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR 
               
               
                   
               
               
                  181 
                 ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN 
               
               
                   
               
               
                  241 
                 GKDPETGEPL DDENIRYQII TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV 
               
               
                   
               
               
                  301 
                 DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ 
               
               
                   
               
               
                  361 
                 LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK 
               
               
                   
               
               
                  421 
                 HFDFEDHTNY ELDIKETLTL KPEGFVVKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN 
               
               
                   
               
               
                  481 
                 TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH 
               
               
                   
               
               
                  541 
                 PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD 
               
               
                   
               
               
                  601 
                 RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH 
               
               
                   
               
               
                  661 
                 GAFSTNVVAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG 
               
               
                   
               
               
                  721 
                 LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE 
               
               
                   
               
               
                  781 
                 LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE 
               
               
                   
               
               
                  841 
                 KQASITVSVV SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI 
               
               
                   
               
               
                  901 
                 MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGIIT 
               
               
                   
               
               
                  961 
                 LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD 
               
               
                   
               
               
                 1021 
                 VHQVSEADAR LWLQQLEEKG RYAKDVWAG 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP102A5 
                   
               
               
                   B. cereus  ATCC14579 
               
               
                 GenBank Accession No. AAP10153 
               
               
                 &gt;gi|29896875|gb|AAP10153.1| NADPH-cytochrome P450 reductase  
               
               
                 [ Bacillus cereus  ATCC 14579] 
               
               
                 SEQ ID NO: 28 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MEKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKIAEEYG PIFQIQTLSD TIIVVSGHEL 
                   
               
               
                   
               
               
                   61 
                 VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETHEPNWK KAHNILMPTF SQRAMKDYHA 
               
               
                   
               
               
                  121 
                 MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR ETPHPFITSM 
               
               
                   
               
               
                  181 
                 TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSG DQEENDLLSR 
               
               
                   
               
               
                  241 
                 MLNVPDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK LKKAYEEVDR 
               
               
                   
               
               
                  301 
                 VLTDPTPTYQ QVMKLKYMRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP IKKGEDRISV 
               
               
                   
               
               
                  361 
                 LIPQLHRDKD AWGDNVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF ALHEATLVMG 
               
               
                   
               
               
                  421 
                 MLLQHFELID YQNYQLDVKQ TLTLKPGDFK IRILPRKQTI SHPTVLAPTE DKLKNDEIKQ 
               
               
                   
               
               
                  481 
                 HVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEVVAL NDRIGSLPKE 
               
               
                   
               
               
                  541 
                 GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS TYQRIPRYID 
               
               
                   
               
               
                  601 
                 EQMAQKGATR FSKRGEADAS GDFEEQLEQW KQNMWSDAMK AFGLELNKNM EKERSTLSLQ 
               
               
                   
               
               
                  661 
                 FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSDRSTRHIE VSLPEGATYK EGDHLGVLPV 
               
               
                   
               
               
                  721 
                 NSEKNINRIL KRFGLNGKDQ VILSASGRSI NHIPLDSPVS LLALLSYSVE VQEAATRAQI 
               
               
                   
               
               
                  781 
                 REMVTFTACP PHKKELEALL EEGVYHEQIL KKRISMLDLL EKYEACEIRF ERFLELLPAL 
               
               
                   
               
               
                  841 
                 KPRYYSISSS PLVAHNRLSI TVGVVNAPAW SGEGTYEGVA SNYLAQRHNK DEIICFIRTP 
               
               
                   
               
               
                  901 
                 QSNFELPKDP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MNLGQAHLYF GCRHPEKDYL 
               
               
                   
               
               
                  961 
                 YRTELENDER DGLISLHTAF SRLEGHPKTY VQHLIKQDRI NLISLLDNGA HLYICGDGSK 
               
               
                   
               
               
                 1021 
                 MAPDVEDTLC QAYQEIHEVS EQEARNWLDR VQDEGRYGKD VWAGI 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP102A7 
                   
               
               
                   B. licheniformis  ATTC1458 
               
               
                 GenBank Accession No. YP 079990 
               
               
                 &gt;gi|52081199|ref|YP_079990.11 cytochrome P450/NADPH-ferrihemoprotein 
               
               
                 reductase [ Bacillus licheniformis  DSM 13 = ATCC 14580] 
               
               
                 SEQ ID NO: 29 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MNKLDGIPIP KTYGPLGNLP LLDKNRVSQS LWKIADEMGP IFQFKFADAI GVFVSSHELV 
                   
               
               
                   
               
               
                   61 
                 KEVSEESRFD KNMGKGLLKV REFSGDGLFT SWTEEPNWRK AHNILLPSFS QKAMKGYHPM 
               
               
                   
               
               
                  121 
                 MQDIAVQLIQ KWSRLNQDES IDVPDDMTRL TLDTIGLCGF NYRFNSFYRE GQHPFIESMV 
               
               
                   
               
               
                  181 
                 RGLSEAMRQT KRFPLQDKLM IQTKRRFNSD VESMFSLVDR IIADRKQAES ESGNDLLSLM 
               
               
                   
               
               
                  241 
                 LHAKDPETGE KLDDENIRYQ IITFLIAGHE TTSGLLSFAI YLLLKHPDKL KKAYEEADRV 
               
               
                   
               
               
                  301 
                 LTDPVPSYKQ VQQLKYIRMI LNESIRLWPT APAFSLYAKE ETVIGGKYLI PKGQSVTVLI 
               
               
                   
               
               
                  361 
                 PKLHRDQSVW GEDAEAFRPE RFEQMDSIPA HAYKPFGNGQ RACIGMQFAL HEATLVLGMI 
               
               
                   
               
               
                  421 
                 LQYFDLEDHA NYQLKIKESL TLKPDGFTIR VRPRKKEAMT AMPGAQPEEN GRQEERPSAP 
               
               
                   
               
               
                  481 
                 AAENTHGTPL LVLYGSNLGT AEEIAKELAE EAREQGFHSR TAELDQYAGA IPAEGAVIIV 
               
               
                   
               
               
                  541 
                 TASYNGNPPD CAKEFVNWLE HDQTDDLRGV KYAVFGCGNR SWASTYQRIP RLIDSVLEKK 
               
               
                   
               
               
                  601 
                 GAQRLHKLGE GDAGDDFEGQ FESWKYDLWP LLRTEFSLAE PEPNQTETDR QALSVEFVNA 
               
               
                   
               
               
                  661 
                 PAASPLAKAY QVFTAKISAN RELQCEKSGR STRHIEISLP EGAAYQEGDH LGVLPQNSEV 
               
               
                   
               
               
                  721 
                 LIGRVFQRFG LNGNEQILIS GRNQASHLPL ERPVHVKDLF QHCVELQEPA TRAQIRELAA 
               
               
                   
               
               
                  781 
                 HTVCPPHQRE LEDLLKDDVY KDQVLNKRLT MLDLLEQYPA CELPFARFLA LLPPLKPRYY 
               
               
                   
               
               
                  841 
                 SISSSPQLNP RQTSITVSVV SGPALSGRGH YKGVASNYLA GLEPGDAISC FIREPQSGFR 
               
               
                   
               
               
                  901 
                 LPEDPETPVI MVGPGTGIAP YRGFLQARRI QRDAGVKLGE AHLYFGCRRP NEDFLYRDEL 
               
               
                   
               
               
                  961 
                 EQAEKDGIVH LHTAFSRLEG RPKTYVQDLL REDAALLIHL LNEGGRLYVC GDGSRMAPAV 
               
               
                   
               
               
                 1021 
                 EQALCEAYRI VQGASREESQ SWLSALLEEG RYAKDVWDGG VSQHNVKADC IART 
               
               
                   
               
            
           
           
               
               
            
               
                 CYPX 
                   
               
               
                 
                   B. thuringiensis serovar konkukian 
                 
               
               
                 str.97-27 
               
               
                 GenBank Accession No. YP 037304 
               
               
                 &gt;gi|49480099|ref|YP_037304.11 NADPH-cytochrome P450 reductase  
               
               
                 [ Bacillus thuringiensis serovar konkukian  str. 97-27] 
               
               
                 SEQ ID NO: 30 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MDKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKLAEEYG PIFQIQTLSD TIIVVSGHEL 
                   
               
               
                   
               
               
                   61 
                 VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETDEPNWK KAHNILMPTF SQRAMKDYHA 
               
               
                   
               
               
                  121 
                 MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR ETPHPFITSM 
               
               
                   
               
               
                  181 
                 TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSE NQEENDLLSR 
               
               
                   
               
               
                  241 
                 MLNVQDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK LKKAYEEVDR 
               
               
                   
               
               
                  301 
                 VLTDSTPTYQ QVMKLKYIRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP IKKGEDRISV 
               
               
                   
               
               
                  361 
                 LIPQLHRDKD AWGDDVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF ALHEATLVMG 
               
               
                   
               
               
                  421 
                 MLLQHFEFID YEDYQLDVKQ TLTLKPGDFK IRIVPRNQTI SHTTVLAPTE EKLKKHEIKK 
               
               
                   
               
               
                  481 
                 QVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEVVAL NDRIGSLPKE 
               
               
                   
               
               
                  541 
                 GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS TYQRIPRYID 
               
               
                   
               
               
                  601 
                 EQMAQKGATR FSTRGEADAS GDFEEQLEQW KQSMWSDAMK AFGLELNKNM EKERSTLSLQ 
               
               
                   
               
               
                  661 
                 FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSERSTRHIE ISLPEGATYK EGDHLGVLPI 
               
               
                   
               
               
                  721 
                 NNEKNVNRIL KRFGLNGKDQ VILSASGRSV NHIPLDSPVR LYDLLSYSVE VQEAATRAQI 
               
               
                   
               
               
                  781 
                 REMVTFTACP PHKKELESLL EDGVYQEQIL KKRISMLDLL EKYEACEIRF ERFLELLPAL 
               
               
                   
               
               
                  841 
                 KPRYYSISSS PLVAQDRLSI TVGVVNAPAW SGEGTYEGVA SNYLAQRHNK DEIICFIRTP 
               
               
                   
               
               
                  901 
                 QSNFQLPENP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MKVGEAHLYF GCRHPEKDYL 
               
               
                   
               
               
                  961 
                 YRTELENDER DGLISLHTAF SRLEGHPKTY VQHVIKEDRI HLISLLDNGA HLYICGDGSK 
               
               
                   
               
               
                 1021 
                 MAPDVEDTLC QAYQEIHEVS EQEARNWLDR LQEEGRYGKD VWAGI 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP102E1 
                   
               
               
                   R. metallidurans  CH34 
               
               
                 GenBank Accession No. YP 585608 
               
               
                 &gt;gi|94312398|ref|YP_585608.1| putative bifunctional P-450:NADPH-P450 
               
               
                 reductase 2 [ Cupriavidus metallidurans  CH34] 
               
               
                 SEQ ID NO: 31 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MSTATPAAAL EPIPRDPGWP IFGNLFQITP GEVGQHLLAR SRHHDGIFEL DFAGKRVPFV 
                   
               
               
                   
               
               
                   61 
                 SSVALASELC DATRFRKIIG PPLSYLRDMA GDGLFTAHSD EPNWGCAHRI LMPAFSQRAM 
               
               
                   
               
               
                  121 
                 KAYFDVMLRV ANRLVDKWDR QGPDADIAVA DDMTRLTLDT IALAGFGYDF ASFASDELDP 
               
               
                   
               
               
                  181 
                 FVMAMVGALG EAMQKLTRLP IQDRFMGRAH RQAAEDIAYM RNLVDDVIRQ RRVSPTSGMD 
               
               
                   
               
               
                  241 
                 LLNLMLEARD PETDRRLDDA NIRNQVITFL IAGHETTSGL LTFALYELLR NPGVLAQAYA 
               
               
                   
               
               
                  301 
                 EVDTVLPGDA LPVYADLARM PVLDRVLKET LRLWPTAPAF AVAPFDDVVL GGRYRLRKDR 
               
               
                   
               
               
                  361 
                 RISVVLTALH RDPKVWANPE RFDIDRFLPE NEAKLPAHAY MPFGQGERAC IGRQFALTEA 
               
               
                   
               
               
                  421 
                 KLALALMLRN FAFQDPHDYQ FRLKETLTIK PDQFVLRVRR RRPHERFVTR QASQAVADAA 
               
               
                   
               
               
                  481 
                 QTDVRGHGQA MTVLCASSLG TARELAEQIH AGAIAAGFDA KLADLDDAVG VLPTSGLVVV 
               
               
                   
               
               
                  541 
                 VAATYNGRAP DSARKFEAML DADDASGYRA NGMRLALLGC GNSQWATYQA FPRRVFDFFI 
               
               
                   
               
               
                  601 
                 TAGAVPLLPR GEADGNGDFD QAAERWLAQL WQALQADGAG TGGLGVDVQV RSMAAIRAET 
               
               
                   
               
               
                  661 
                 LPAGTQAFTV LSNDELVGDP SGLWDFSIEA PRTSTRDIRL QLPPGITYRT GDHIAVWPQN 
               
               
                   
               
               
                  721 
                 DAQLVSELCE RLDLDPDAQA TISAPHGMGR GLPIDQALPV RQLLTHFIEL QDVVSRQTLR 
               
               
                   
               
               
                  781 
                 ALAQATRCPF TKQSIEQLAS DDAEHGYATK VVARRLGILD VLVEHPAIAL TLQELLACTV 
               
               
                   
               
               
                  841 
                 PMRPRLYSIA SSPLVSPDVA TLLVGTVCAP ALSGRGQFRG VASTWLQHLP PGARVSASIR 
               
               
                   
               
               
                  901 
                 TPNPPFAPDP DPAAPMLLIG PGTGIAPFRG FLEERALRKM AGNAVTPAQL YFGCRHPQHD 
               
               
                   
               
               
                  961 
                 WLYREDIERW AGQGVVEVHP AYSVVPDAPR YVQDLLWQRR EQVWAQVRDG ATIYVCGDGR 
               
               
                   
               
               
                 1021 
                 RMAPAVRQTL IEIGMAQGGM TDKAASDWFG GLVAQGRYRQ DVFN 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505X 
                   
               
               
                   A. fumigatus  Af293 
               
               
                 GenBank Accession No. EAL92660 
               
               
                 &gt;gi|66852335|gb|EAL92660.1| P450 family fatty acid hydroxylase, putative 
               
               
                 [ Aspergillus fumigatus  Af293] 
               
               
                 SEQ ID NO: 32 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MSESKTVPIP GPRGVPLLGN IYDIEQEVPL RSINLMADQY GPIYRLTTFG WSRVFVSTHE 
                   
               
               
                   
               
               
                   61 
                 LVDEVCDEER FTKVVTAGLN QIRNGVHDGL FTANFPGEEN WAIAHRVLVP AFGPLSIRGM 
               
               
                   
               
               
                  121 
                 FDEMYDIATQ LVMKWARHGP TVPIMVTDDF TRLTLDTIAL CAMGTRFNSF YHEEMHPFVE 
               
               
                   
               
               
                  181 
                 AMVGLLQGSG DRARRPALLN NLPTSENSKY WDDIAFLRNL AQELVEARRK NPEDKKDLLN 
               
               
                   
               
               
                  241 
                 ALILGRDPKT GKGLTDESII DNMITFLIAG HETTSGLLSF LFYYLLKTPN AYKKAQEEVD 
               
               
                   
               
               
                  301 
                 SVVGRRKITV EDMSRLPYLN AVMRETLRLR STAPLIAVHA HPEKNKEDPV TLGGGKYVLN 
               
               
                   
               
               
                  361 
                 KDEPIVIILD KLHRDPQVYG PDAEEFKPER MLDENFEKLP KNAWKPFGNG MRACIGRPFA 
               
               
                   
               
               
                  421 
                 WQEALLVVAI LLQNFNFQMD DPSYNLHIKQ TLTIKPKDFH MRATLRHGLD ATKLGIALSG 
               
               
                   
               
               
                  481 
                 SADRAPPESS GAASRVRKQA TPPAGQLKPM HIFFGSNTGT CETFARRLAD DAVGYGFAAD 
               
               
                   
               
               
                  541 
                 VQSLDSAMQN VPKDEPVVFI TASYEGQPPD NAAHFFEWLS ALKENELEGV NYAVFGCGHH 
               
               
                   
               
               
                  601 
                 DWQATFHRIP KAVNQLVAEH GGNRLCDLGL ADAANSDMFT DFDSWGESTF WPAITSKFGG 
               
               
                   
               
               
                  661 
                 GKSDEPKPSS SLQVEVSTGM RASTLGLQLQ EGLVIDNQLL SAPDVPAKRM IRFKLPSDMS 
               
               
                   
               
               
                  721 
                 YRCGDYLAVL PVNPTSVVRR AIRRFDLPWD AMLTIRKPSQ APKGSTSIPL DTPISAFELL 
               
               
                   
               
               
                  781 
                 STYVELSQPA SKRDLTALAD AAITDADAQA ELRYLASSPT RFTEEIVKKR MSPLDLLIRY 
               
               
                   
               
               
                  841 
                 PSIKLPVGDF LAMLPPMRVR QYSISSSPLA DPSECSITFS VLNAPALAAA SLPPAERAEA 
               
               
                   
               
               
                  901 
                 EQYMGVASTY LSELKPGERA HIAVRPSHSG FKPPMDLKAP MIMACAGSGL APFRGFIMDR 
               
               
                   
               
               
                  961 
                 AEKIRGRRSS VGADGQLPEV EQPAKAILYV GCRTKGKDDI HATELAEWAQ LGAVDVRWAY 
               
               
                   
               
               
                 1021 
                 SRPEDGSKGR HVQDLMLEDR EELVSLFDQG ARIYVCGSTG VGNGVRQACK DIYLERRRQL 
               
               
                   
               
               
                 1081 
                 RQAARERGEE VPAEEDEDAA AEQFLDNLRT KERYATDVFT 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A8 
                   
               
               
                   A. nidulans  FGSC A4 
               
               
                 GenBank Accession No. EAA58234 
               
               
                 &gt;gi|40739044|gb|EAA58234.1| hypothetical protein AN6835.2  
               
               
                 [ Aspergillus   nidulans  FGSC A4] 
               
               
                 SEQ ID NO: 33 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MAEIPEPKGL PLIGNIGTID QEFPLGSMVA LAEEHGEIYR LRFPGRTVVV VSTHALVNET 
                   
               
               
                   
               
               
                   61 
                 CDEKRFRKSV NSALAHVREG VHDGLFTAKM GEVNWEIAHR VLMPAFGPLS IRGMFDEMHD 
               
               
                   
               
               
                  121 
                 IASQLALKWA RYGPDCPIMV TDDFTRLTLD TLALCSMGYR FNSYYSPVLH PFIEAMGDFL 
               
               
                   
               
               
                  181 
                 TEAGEKPRRP PLPAVFFRNR DQKFQDDIAV LRDTAQGVLQ ARKEGKSDRN DLLSAMLRGV 
               
               
                   
               
               
                  241 
                 DSQTGQKMTD ESIMDNLITF LIAGHETTSG LLSFVFYQLL KHPETYRTAQ QEVDNVVGQG 
               
               
                   
               
               
                  301 
                 VIEVSHLSKL PYINSVLRET LRLNATIPLF TVEAFEDTLL AGKYPVKAGE TIVNLLAKSH 
               
               
                   
               
               
                  361 
                 LDPEVYGEDA LEFKPERMSD ELFNARLKQF PSAWKPFGNG MRACIGRPFA WQEALLVMAM 
               
               
                   
               
               
                  421 
                 LLQNFDFSLA DPNYDLKFKQ TLTIKPKDMF MKARLRHGLT PTTLERRLAG LAVESATQDK 
               
               
                   
               
               
                  481 
                 IVTNPADNSV TGTRLTILYG SNSGTCETLA RRIAADAPSK GFHVMRFDGL DSGRSALPTD 
               
               
                   
               
               
                  541 
                 HPVVIVTSSY EGQPPENAKQ FVSWLEELEQ QNESLQLKGV DFAVFGCFKE WAQTFHRIPK 
               
               
                   
               
               
                  601 
                 LVDSLLEKLG GSRLTDLGLA DVSTDELFST FETWADDVLW PRLVAQYGAD GKTQAHGSSA 
               
               
                   
               
               
                  661 
                 GHEAASNAAV EVTVSNSRTQ ALRQDVGQAM VVETRLLTAE SEKERRKKHL EIRLPDGVSY 
               
               
                   
               
               
                  721 
                 TAGDYLAVLP INPPETVRRA MRQFKLSWDA QITIAPSGPT TALPTDGPIA ANDIFSTYVE 
               
               
                   
               
               
                  781 
                 LSQPATRKDL RIMADATTDP DVQKILRTYA NETYTAEILT KSISVLDILE QHPAIDLPLG 
               
               
                   
               
               
                  841 
                 TFLLMLPSMR MRQYSISSSP LLTPTTATIT ISVLDAPSRS RSNGSRHLGV ATSYLDSLSV 
               
               
                   
               
               
                  901 
                 GDHLQVTVRK NPSSGFRLPS EPETTPMICI AAGSGIAPFR AFLQERAVMM EQDKDRKLAP 
               
               
                   
               
               
                  961 
                 ALLFFGCRAP GIDDLYREQL EEWQARGVVD ARWAFSRQSD DTKGCRHVDD RILADREDVV 
               
               
                   
               
               
                 1021 
                 KLWRDGARVY VCGSGALAQS VRSAMVTVLR DEMETTGDGS DNGKAEKWFD EQRNVRYVMD 
               
               
                   
               
               
                 1081 
                 VFD 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A3 
                   
               
               
                   A. oryzae  ATCC42149 
               
               
                 Uniprot Accession No. Q2U4F1 
               
               
                 &gt;gi|121928062|sp|Q2U4F1|Q2U4F1_ASPOR Cytochrome P450 
               
               
                 SEQ ID NO: 34 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MRQNDNEKQI CPIPGPQGLP FLGNILDIDL DNGTMSTLKI AKTYYPIFKF TFAGETSIVI 
                   
               
               
                   
               
               
                   61 
                 NSVALLSELC DETRFHKHVS FGLELLRSGT HDGLFTAYDH EKNWELAHRL LVPAFGPLRI 
               
               
                   
               
               
                  121 
                 REMFPQMHDI AQQLCLKWQR YGPRRPLNLV DDFTRTTLDT IALCAMGYRF NSFYSEGDFH 
               
               
                   
               
               
                  181 
                 PFIKSMVRFL KEAETQATLP SFISNLRVRA KRRTQLDIDL MRTVCREIVT ERRQTNLDHK 
               
               
                   
               
               
                  241 
                 NDLLDTMLTS RDSLSGDALS DESIIDNILT FLVAGHETTS GLLSFAVYYL LTTPDAMAKA 
               
               
                   
               
               
                  301 
                 AHEVDDVVGD QELTIEHLSM LKYLNAILRE TLRLMPTAPG FSVTPYKPEI IGGKYEVKPG 
               
               
                   
               
               
                  361 
                 DSLDVFLAAV HRDPAVYGSD ADEFRPERMS DEHFQKLPAN SWKPFGNGKR SCIGRAFAWQ 
               
               
                   
               
               
                  421 
                 EALMILALIL QSFSLNLVDR GYTLKLKESL TIKPDNLWAY ATPRPGRNVL HTRLALQTNS 
               
               
                   
               
               
                  481 
                 THPEGLMSLK HETVESQPAT ILYGSNSGTC EALAHRLAIE MSSKGRFVCK VQPMDAIEHR 
               
               
                   
               
               
                  541 
                 RLPRGQPVII ITGSYDGRPP ENARHFVKWL QSLKGNDLEG IQYAVFGCGL PGHHDWSTTF 
               
               
                   
               
               
                  601 
                 YKIPTLIDTI MAEHGGARLA PRGSADTAED DPFAELESWS ERSVWPGLEA AFDLVRHNSS 
               
               
                   
               
               
                  661 
                 DGTGKSTRIT IRSPYTLRAA HETAVVHQVR VLTSAETTKK VHVELALPDT INYRPGDHLA 
               
               
                   
               
               
                  721 
                 ILPLNSRQSV QRVLSLFQIG SDTILYMTSS SATSLPTDTP ISAHDLLSGY VELNQVATPT 
               
               
                   
               
               
                  781 
                 SLRSLAAKAT DEKTAEYLEA LATDRYTTEV RGNHLSLLDI LESYSVPSIE IQHYIQMLPL 
               
               
                   
               
               
                  841 
                 LRPRQYTISS SPRLNRGQAS LTVSVMERAD VGGPRNCAGV ASNYLASCTP GSILRVSLRQ 
               
               
                   
               
               
                  901 
                 ANPDFRLPDE SCSHPIIMVA AGSGIAPFRA FVQERSVRQK EGIILPPAFL FFGCRRADLD 
               
               
                   
               
               
                  961 
                 DLYREELDAF EEQGVVTLFR AFSRAQSESH GCKYVQDLLW MERVRVKTLW GQDAKVFVCG 
               
               
                   
               
               
                 1021 
                 SVRMNEGVKA IISKIVSPTP TEELARRYIA ETFI 
               
               
                   
               
            
           
           
               
               
            
               
                 CYPX 
                   
               
               
                   A. oryzae  ATCC42149 
               
               
                 Uniprot Accession No. Q2UNA2 
               
               
                 &gt;gi|121938553|sp|Q2UNA2|Q2UNA2_ASPOR Cytochrome P450 
               
               
                 SEQ ID NO: 35 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MSTPKAEPVP IPGPRGVPLM GNILDIESEI PLRSLEMMAD TYGPIYRLTT FGFSRCMISS 
                   
               
               
                   
               
               
                   61 
                 HELAAEVFDE ERFTKKIMAG LSELRHGIHD GLFTAHMGEE NWEIAHRVLM PAFGPLNIQN 
               
               
                   
               
               
                  121 
                 MFDEMHDIAT QLVMKWARQG PKQKIMVTDD FTRLTLDTIA LCAMGTRFNS FYSEEMHPFV 
               
               
                   
               
               
                  181 
                 DAMVGMLKTA GDRSRRPGLV NNLPTTENNK YWEDIDYLRN LCKELVDTRK KNPTDKKDLL 
               
               
                   
               
               
                  241 
                 NALINGRDPK TGKGMSYDSI IDNMITFLIA GHETTSGSLS FAFYNMLKNP QAYQKAQEEV 
               
               
                   
               
               
                  301 
                 DRVIGRRRIT VEDLQKLPYI TAVMRETLRL TPTAPAIAVG PHPTKNHEDP VTLGNGKYVL 
               
               
                   
               
               
                  361 
                 GKDEPCALLL GKIQRDPKVY GPDAEEFKPE RMLDEHFNKL PKHAWKPFGN GMRACIGRPF 
               
               
                   
               
               
                  421 
                 AWQEALLVIA MLLQNFNFQM DDPSYNIQLK QTLTIKPNHF YMRAALREGL DAVHLGSALS 
               
               
                   
               
               
                  481 
                 ASSSEHADHA AGHGKAGAAK KGADLKPMHV YYGSNTGTCE AFARRLADDA TSYGYSAEVE 
               
               
                   
               
               
                  541 
                 SLDSAKDSIP KNGPVVFITA SYEGQPPDNA AHFFEWLSAL KGDKPLDGVN YAVFGCGHHD 
               
               
                   
               
               
                  601 
                 WQTTFYRIPK EVNRLVGENG ANRLCEIGLA DTANADIVTD FDTWGETSFW PAVAAKFGSN 
               
               
                   
               
               
                  661 
                 TQGSQKSSTF RVEVSSGHRA TTLGLQLQEG LVVENTLLTQ AGVPAKRTIR FKLPTDTQYK 
               
               
                   
               
               
                  721 
                 CGDYLAILPV NPSTVVRKVM SRFDLPWDAV LRIEKASPSS SKHISIPMDT QVSAYDLFAT 
               
               
                   
               
               
                  781 
                 YVELSQPASK RDLAVLADAA AVDPETQAEL QAIASDPARF AEISQKRISV LDLLLQYPSI 
               
               
                   
               
               
                  841 
                 NLAIGDFVAM LPPMRVRQYS ISSSPLVDPT ECSITFSVLK APSLAALTKE DEYLGVASTY 
               
               
                   
               
               
                  901 
                 LSELRSGERV QLSVRPSHTG FKPPTELSTP MIMACAGSGL APFRGFVMDR AEKIRGRRSS 
               
               
                   
               
               
                  961 
                 GSMPEQPAKA ILYAGCRTQG KDDIHADELA EWEKIGAVEV RRAYSRPSDG SKGTHVQDLM 
               
               
                   
               
               
                 1021 
                 MEDKKELIDL FESGARIYVC GTPGVGNAVR DSIKSMFLER REEIRRIAKE KGEPVSDDDE 
               
               
                   
               
               
                 1081 
                 ETAFEKFLDD MKTKERYTTD IFA 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A1 
                   
               
               
                 
                   F. oxysporum 
                 
               
               
                 Uniprot Accession No. Q9Y8G7 
               
               
                 &gt;gi|22653677|sp|Q9Y8G7.1|C505_FUSOX RecName: Full = Bifunctional  
               
               
                 P-450:NADPH-P450 reductase; AltName: Full = Cytochrome P450foxy;  
               
               
                 AltName: Full = Fatty acid omega-hydroxylase; Includes: RecName:  
               
               
                 Full = Cytochrome P450 505; Includes: 
               
               
                 RecName: Full = NADPH--cytochrome P450 reductase 
               
               
                 SEQ ID NO: 36 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 maesvpipep pgyplignlg eftsnplsdl nrladtygpi frlrlgakap ifvssnslin 
                   
               
               
                   
               
               
                   61 
                 evcdekrfkk tlksvlsqvr egvhdglfta fedepnwgka hrilvpafgp lsirgmfpem 
               
               
                   
               
               
                  121 
                 hdiatqlcmk farhgprtpi dtsdnftrla ldtlalcamd frfysyykee lhpfieamgd 
               
               
                   
               
               
                  181 
                 fltesgnrnr rppfapnfly raanekfygd ialmksvade vvaarkasps drkdllaaml 
               
               
                   
               
               
                  241 
                 ngvdpqtgek lsdenitnql itfliaghet tsgtlsfamy qllknpeays kvqkevdevv 
               
               
                   
               
               
                  301 
                 grgpvlvehl tklpyisavl retlrinspi tafgleaidd tflggkylvk kgeivtalls 
               
               
                   
               
               
                  361 
                 rghvdpvvyg ndadkfiper mlddefarin keypncwkpf gngkracigr pfawqeslla 
               
               
                   
               
               
                  421 
                 mvvlfqnfnf tmtdpnyale ikqtltikpd hfyinatlrh gmtptelehv lagngatsss 
               
               
                   
               
               
                  481 
                 thnikaaanl dakagsgkpm aifygsnsgt cealanrlas dapshgfsat tvgpldqakq 
               
               
                   
               
               
                  541 
                 nlpedrpvvi vtasyeggpp snaahfikwm edldgndmek vsyavfacgh hdwvetfhri 
               
               
                   
               
               
                  601 
                 pklvdstlek rggtrlvpmg sadaatsdmf sdfeawediv lwpglkekyk isdeesggqk 
               
               
                   
               
               
                  661 
                 gllvevstpr ktslrqdvee alvvaektlt ksgpakkhie iqlpsamtyk agdylailpl 
               
               
                   
               
               
                  721 
                 npkstvarvf rrfslawdsf lkiqsegptt lptnvaisaf dvfsayvels qpatkrnila 
               
               
                   
               
               
                  781 
                 laeatedkdt iqelerlagd ayqaeispkr vsvldllekf pavalpissy lamlppmrvr 
               
               
                   
               
               
                  841 
                 gysissspfa dpskltltys lldapslsgq grhvgvatnf lshltagdkl hvsvrassea 
               
               
                   
               
               
                  901 
                 fhlpsdaekt piicvaagtg laplrgfiqe raamlaagrt lapallffgc rnpeiddlya 
               
               
                   
               
               
                  961 
                 eeferwekmg avdvrraysr atdksegcky vqdrvyhdra dvfkvwdqga kvficgsrei 
               
               
                   
               
               
                 1021 
                 gkavedvcvr laiekaqqng rdvteemara wfersrnerf atdvfd 
               
               
                   
               
            
           
           
               
               
            
               
                 CYPX 
                   
               
               
                 
                   G. moniliformis 
                 
               
               
                 GenBank Accession No. AAG27132 
               
               
                 &gt;gi|11035011|gb|AAG27132.11 Fum6p [ Fusarium verticillioides ] 
               
               
                 SEQ ID NO: 37 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MSATALFTRR SVSTSNPELR PIPGPKPLPL LGNLFDFDFD NLTKSLGELG KIHGPIYSIT 
                   
               
               
                   
               
               
                   61 
                 FGASTEIMVT SREIAQELCD ETRFCKLPGG ALDVMKAVVG DGLFTAETSN PKWAIAHRII 
               
               
                   
               
               
                  121 
                 TPLFGAMRIR GMFDDMKDIC EQMCLRWARF GPDEPLNVCD NMTKLTLDTI ALCTIDYRFN 
               
               
                   
               
               
                  181 
                 SFYRENGAAH PFAEAVVDVM TESFDQSNLP DFVNNYVRFR AMAKFKRQAA ELRRQTEELI 
               
               
                   
               
               
                  241 
                 AARRQNPVDR DDLLNAMLSA KDPKTGEGLS PESIVDNLLT FLIAGHETTS SLLSFCFYYL 
               
               
                   
               
               
                  301 
                 LENPHVLRRV QQEVDTVVGS DTITVDHLSS MPYLEAVLRE TLRLRDPGPG FYVKPLKDEV 
               
               
                   
               
               
                  361 
                 VAGKYAVNKD QPLFIVFDSV HRDQSTYGAD ADEFRPERML KDGFDKLPPC AWKPFGNGVR 
               
               
                   
               
               
                  421 
                 ACVGRPFAMQ QAILAVAMVL HKFDLVKDES YTLKYHVTMT VRPVGFTMKV RLRQGQRATD 
               
               
                   
               
               
                  481 
                 LAMGLHRGHS QEASAAASPS RASLKRLSSD VNGDDTDHKS QIAVLYASNS GSCEALAYRL 
               
               
                   
               
               
                  541 
                 AAEATERGFG IRAVDVVNNA IDRIPVGSPV ILITASYNGE PADDAQEFVP WLKSLESGRL 
               
               
                   
               
               
                  601 
                 NGVKFAVFGN GHRDWANTLF AVPRLIDSEL ARCGAERVSL MGVSDTCDSS DPFSDFERWI 
               
               
                   
               
               
                  661 
                 DEKLFPELET PHGPGGVKNG DRAVPRQELQ VSLGQPPRIT MRKGYVRAIV TEARSLSSPG 
               
               
                   
               
               
                  721 
                 VPEKRHLELL LPKDFNYKAG DHVYILPRNS PRDVVRALSY FGLGEDTLIT IRNTARKLSL 
               
               
                   
               
               
                  781 
                 GLPLDTPITA TDLLGAYVEL GRTASLKNLW TLVDAAGHGS RAALLSLTEP ERFRAEVQDR 
               
               
                   
               
               
                  841 
                 HVSILDLLER FPDIDLSLSC FLPMLAQIRP RAYSFSSAPD WKPGHATLTY TVVDFATPAT 
               
               
                   
               
               
                  901 
                 QGINGSSKSK AVGDGTAVVQ RQGLASSYLS SLGPGTSLYV SLHRASPYFC LQKSTSLPVI 
               
               
                   
               
               
                  961 
                 MVGAGTGLAP FRAFLQERRM AAEGAKQRFG PALLFFGCRG PRLDSLYSVE LEAYETIGLV 
               
               
                   
               
               
                 1021 
                 QVRRAYSRDP SAQDAQGCKY VTDRLGKCRD EVARLWMDGA QVLVCGGKKM ANDVLEVLGP 
               
               
                   
               
               
                 1081 
                 MLLEIDQKRG ETTAKTVVEW RARLDKSRYV EEVYV 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A7 
                   
               
               
                   G. zeae  PH1 
               
               
                 GenBank Accession No. EAA67736 
               
               
                 &gt;gi|42544893|gb|EAA67736.1| C505_FUSOX Bifunctional P-450:NADPH-P450 
               
               
                 reductase (Fatty acid omega-hydroxylase) (P450foxy)  
               
               
                 [ Gibberella zeae  PH-1] 
               
               
                 SEQ ID NO: 38 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MAESVPIPEP PGYPLIGNLG EFKTNPLNDL NRLADTYGPI FRLHLGSKTP TFVSSNAFIN 
                   
               
               
                   
               
               
                   61 
                 EVCDEKRFKK TLKSVLSVVR EGVHDGLFTA FEDEPNWGKA HRILIPAFGP LSIRNMFPEM 
               
               
                   
               
               
                  121 
                 HEIANQLCMK LARHGPHTPV DASDNFTRLA LDTLALCAMD FRFNSYYKEE LHPFIEAMGD 
               
               
                   
               
               
                  181 
                 FLLESGNRNR RPAFAPNFLY RAANDKFYAD IALMKSVADE VVATRKQNPT DRKDLLAAML 
               
               
                   
               
               
                  241 
                 EGVDPQTGEK LSDDNITNQL ITFLIAGHET TSGTLSFAMY HLLKNPEAYN KLQKEIDEVI 
               
               
                   
               
               
                  301 
                 GRDPVTVEHL TKLPYLSAVL RETLRISSPI TGFGVEAIED TFLGGKYLIK KGETVLSVLS 
               
               
                   
               
               
                  361 
                 RGHVDPVVYG PDAEKFVPER MLDDEFARLN KEFPNCWKPF GNGKRACIGR PFAWQESLLA 
               
               
                   
               
               
                  421 
                 MALLFQNFNF TQTDPNYELQ IKQNLTIKPD NFFFNCTLRH GMTPTDLEGQ LAGKGATTSI 
               
               
                   
               
               
                  481 
                 ASHIKAPAAS KGAKASNGKP MAIYYGSNSG TCEALANRLA SDAAGHGFSA SVIGTLDQAK 
               
               
                   
               
               
                  541 
                 QNLPEDRPVV IVTASYEGQP PSNAAHFIKW MEDLAGNEME KVSYAVFGCG HHDWVDTFLR 
               
               
                   
               
               
                  601 
                 IPKLVDTTLE QRGGTRLVPM GSADAATSDM FSDFEAWEDT VLWPSLKEKY NVTDDEASGQ 
               
               
                   
               
               
                  661 
                 RGLLVEVTTP RKTTLRQDVE EALVVSEKTL TKTGPAKKHI EIQLPSGMTY KAGDYLAILP 
               
               
                   
               
               
                  721 
                 LNPRKTVSRV FRRFSLAWDS FLKIQSDGPT TLPINIAISA FDVFSAYVEL SQPATKRNIL 
               
               
                   
               
               
                  781 
                 ALSEATEDKA TIQELEKLAG DAYQEDVSAK KVSVLDLLEK YPAVALPISS YLAMLPPMRV 
               
               
                   
               
               
                  841 
                 RQYSISSSPF ADPSKLTLTY SLLDAPSLSG QGRHVGVATN FLSQLIAGDK LHISVRASSA 
               
               
                   
               
               
                  901 
                 AFHLPSDPET TPIICVAAGT GLAPFRGFIQ ERAAMLAAGR KLAPALLFFG CRDPENDDLY 
               
               
                   
               
               
                  961 
                 AEELARWEQM GAVDVRRAYS RATDKSEGCK YVQDRIYHDR ADVFKVWDQG AKVFICGSRE 
               
               
                   
               
               
                 1021 
                 IGKAVEDICV RLAMERSEAT QEGKGATEEK AREWFERSRN ERFATDVFD 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505C2 
                   
               
               
                   G. zeae  PH1a 
               
               
                 GenBank Accession No. EAA77183 
               
               
                 &gt;gi|42554340|gb|EAA77183.1| hypothetical protein FG07596.1  
               
               
                 [ Gibberella zeae  PH-1] 
               
               
                 SEQ ID NO: 39 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MAIKDGGKKS GQIPGPKGLP VLGNLFDLDL SDSLTSLINI GQKYAPIFSL ELGGHREVMI 
                   
               
               
                   
               
               
                   61 
                 CSRDLLDELC DETRFHKIVT GGVDKLRPLA GDGLFTAQHG NHDWGIAHRI LMPLFGPLKI 
               
               
                   
               
               
                  121 
                 REMFDDMQDV SEQLCLKWAR LGPSATIDVA NDFTRLTLDT IALCTMGYRF NSFYSNDKMH 
               
               
                   
               
               
                  181 
                 PFVDSMVAAL IDADKQSMFP DFIGACRVKA LSAFRKHAAI MKGTCNELIQ ERRKNPIEGT 
               
               
                   
               
               
                  241 
                 DLLTAMMEGK DPKTGEGMSD DLIVQNLITF LIAGHETTSG LLSFAFYYLL ENPHTLEKAR 
               
               
                   
               
               
                  301 
                 AEVDEVVGDQ ALNVDHLTKM PYVNMILRET LRLMPTAPGF FVTPHKDEII GGKYAVPANE 
               
               
                   
               
               
                  361 
                 SLFCFLHLIH RDPKVWGADA EEFRPERMAD EFFEALPKNA WKPFGNGMRG CIGREFAWQE 
               
               
                   
               
               
                  421 
                 AKLITVMILQ NFELSKADPS YKLKIKQSLT IKPDGFNMHA KLRNDRKVSG LFKAPSLSSQ 
               
               
                   
               
               
                  481 
                 QPSLSSRQSI NAINAKDLKP ISIFYGSNTG TCEALAQKLS ADCVASGFMP SKPLPLDMAT 
               
               
                   
               
               
                  541 
                 KNLSKDGPNI LLAASYDGRP SDNAEEFTKW AESLKPGELE GVQFAVFGCG HKDWVSTYFK 
               
               
                   
               
               
                  601 
                 IPKILDKCLA DAGAERLVEI GLTDASTGRL YSDFDDWENQ KLFTELSKRQ GVTPTDDSHL 
               
               
                   
               
               
                  661 
                 ELNVTVIQPQ NNDMGGNFKR AEVVENTLLT YPGVSRKHSL LLKLPKDMEY TPGDHVLVLP 
               
               
                   
               
               
                  721 
                 KNPPQLVEQA MSCFGVDSDT ALTISSKRPT FLPTDTPILI SSLLSSLVEL SQTVSRTSLK 
               
               
                   
               
               
                  781 
                 RLADFADDDD TKACVERIAG DDYTVEVEEQ RMSLLDILRK YPGINMPLST FLSMLPQMRP 
               
               
                   
               
               
                  841 
                 RTYSFASAPE WKQGHGMLLF SVVEAEEGTV SRPGGLATNY MAQLRQGDSI LVEPRPCRPE 
               
               
                   
               
               
                  901 
                 LRTTMMLPEP KVPIIMIAVG AGLAPFLGYL QKRFLQAQSQ RTALPPCTLL FGCRGAKMDD 
               
               
                   
               
               
                  961 
                 ICRAQLDEYS RAGVVSVHRA YSRDPDSQCK YVQGLVTKHS ETLAKQWAQG AIVMVCSGKK 
               
               
                   
               
               
                 1021 
                 VSDGVMNVLS PILFAEEKRS GMTGADSVDV WRQNVPKERM ILEVFG 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A5 
                   
               
               
                   M. grisea  70-15 syn 
               
               
                 GenBank Accession No. XP 365223 
               
               
                 &gt;gi|145601517|ref|XP_365223.2| hypothetical protein MGG 01925  
               
               
                 [ Magnaporthe   oryzae  70-15] 
               
               
                 SEQ ID NO: 40 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MFFLSSSLAY MAATQSRDWA SFGVSLPSTA LGRHLQAAMP FLSEENHKSQ GTVLIPDAQG 
                   
               
               
                   
               
               
                   61 
                 PIPFLGSVPL VDPELPSQSL QRLARQYGEI YRFVIPGRQS PILVSTHALV NELCDEKRFK 
               
               
                   
               
               
                  121 
                 KKVAAALLGL REAIHDGLFT AHNDEPNWGI AHRILMPAFG PMAIKGMFDE MHDVASQMIL 
               
               
                   
               
               
                  181 
                 KWARHGSTTP IMVSDDFTRL TLDTIALCSM GYRFNSFYHD SMHEFIEAMT CWMKESGNKT 
               
               
                   
               
               
                  241 
                 RRLLPDVFYR TTDKKWHDDA EILRRTADEV LKARKENPSG RKDLLTAMIE GVDPKTGGKL 
               
               
                   
               
               
                  301 
                 SDSSIIDNLI TFLIAGHETT SGMLSFAFYL LLKNPTAYRK AQQEIDDLCG REPITVEHLS 
               
               
                   
               
               
                  361 
                 KMPYITAVLR ETLRLYSTIP AFVVEAIEDT VVGGKYAIPK NHPIFLMIAE SHRDPKVYGD 
               
               
                   
               
               
                  421 
                 DAQEFEPERM LDGQFERRNR EFPNSWKPFG NGMRGCIGRA FAWQEALLIT AMLLQNFNFV 
               
               
                   
               
               
                  481 
                 MHDPAYQLSI KENLTLKPDN FYMRAILRHG MSPTELERSI SGVAPTGNKT PPRNATRTSS 
               
               
                   
               
               
                  541 
                 PDPEDGGIPM SIYYGSNSGT CESLAHKLAV DASAQGFKAE TVDVLDAANQ KLPAGNRGPV 
               
               
                   
               
               
                  601 
                 VLITASYEGL PPDNAKHFVE WLENLKGGDE LVDTSYAVFG CGHQDWTKTF HRIPKLVDEK 
               
               
                   
               
               
                  661 
                 LAEHGAVRLA PLGLSNAAHG DMFVDFETWE FETLWPALAD RYKTGAGRQD AAATDLTAAL 
               
               
                   
               
               
                  721 
                 SQLSVEVSHP RAADLRQDVG EAVVVAARDL TAPGAPPKRH MEIRLPKTGG RVHYSAGDYL 
               
               
                   
               
               
                  781 
                 AVLPVNPKST VERAMRRFGL AWDAHVTIRS GGRTTLPTGA PVSAREVLSS YVELTQPATK 
               
               
                   
               
               
                  841 
                 RGIAVLAGAV TGGPAAEQEQ AKAALLDLAG DSYALEVSAK RVGVLDLLER FPACAVPFGT 
               
               
                   
               
               
                  901 
                 FLALLPPMRV RQYSISSSPL WNDEHATLTY SVLSAPSLAD PARTHVGVAS SYLAGLGEGD 
               
               
                   
               
               
                  961 
                 HLHVALRPSH VAFRLPSPET PVVCVCAGSG MAPFRAFAQE RAALVGAGRK VAPLLLFFGC 
               
               
                   
               
               
                 1021 
                 REPGVDDLYR EELEGWEAKG VLSVRRAYSR RTEQSEGCRY VQDRLLKNRA EVKSLWSQDA 
               
               
                   
               
               
                 1081 
                 KVFVCGSREV AEGVKEAMFK VVAGKEGSSE EVQAWYEEVR NVRYASDIFD 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP505A2 
                   
               
               
                   N. crassa  OR74 A 
               
               
                 GenBank Accession No. XP 961848 
               
               
                 &gt;gi|85104987|ref|XP_961848.1| bifunctional P-450:NADPH-P450 reductase 
               
               
                 [ Neurospora crassa  OR74A] 
               
               
                 SEQ ID NO: 41 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 MSSDETPQTI PIPGPPGLPL VGNSFDIDTE FPLGSMLNFA DQYGEIFRLN FPGRNTVFVT 
                   
               
               
                   
               
               
                   61 
                 SQALVHELCD EKRFQKTVNS ALHEIRHGIH DGLFTARNDE PNWGIAHRIL MPAFGPMAIQ 
               
               
                   
               
               
                  121 
                 NMFPEMHEIA SQLALKWARH GPNQSIKVTD DFTRLTLDTI ALCSMDYRFN SYYHDDMHPF 
               
               
                   
               
               
                  181 
                 IDAMASFLVE SGNRSRRPAL PAFMYSKVDR KFYDDIRVLR ETAEGVLKSR KEHPSERKDL 
               
               
                   
               
               
                  241 
                 LTAMLDGVDP KTGGKLSDDS IIDNLITFLI AGHETTSGLL SFAFVQLLKN PETYRKAQKE 
               
               
                   
               
               
                  301 
                 VDDVCGKGPI KLEHMNKLHY IAAVLRETLR LCPTIPVIGV ESKEDTVIGG KYEVSKGQPF 
               
               
                   
               
               
                  361 
                 ALLFAKSHVD PAVYGDTAND FDPERMLDEN FERLNKEFPD CWKPFGNGMR ACIGRPFAWQ 
               
               
                   
               
               
                  421 
                 EALLVMAVCL QNFNFMPEDP NYTLQYKQTL TTKPKGFYMR AMLRDGMSAL DLERRLKGEL 
               
               
                   
               
               
                  481 
                 VAPKPTAQGP VSGQPKKSGE GKPISIYYGS NTGTCETFAQ RLASDAEAHG FTATIIDSLD 
               
               
                   
               
               
                  541 
                 AANQNLPKDR PVVFITASYE GQPPDNAALF VGWLESLTGN ELEGVQYAVF GCGHHDWAQT 
               
               
                   
               
               
                  601 
                 FHRIPKLVDN TVSERGGDRI CSLGLADAGK GEMFTEFEQW EDEVFWPAME EKYEVSRKED 
               
               
                   
               
               
                  661 
                 DNEALLQSGL TVNFSKPRSS TLRQDVQEAV VVDAKTITAP GAPPKRHIEV QLSSDSGAYR 
               
               
                   
               
               
                  721 
                 SGDYLAVLPI NPKETVNRVM RRFQLAWDTN ITIEASRQTT ILPTGVPMPV HDVLGAYVEL 
               
               
                   
               
               
                  781 
                 SQPATKKNIL ALAEAADNAE TKATLRQLAG PEYTEKITSR RVSILDLLEQ FPSIPLPFSS 
               
               
                   
               
               
                  841 
                 FLSLLPPMRV RQYSISSSPL WNPSHVTLTY SLLESPSLSN PDKKHVGVAT SYLASLEAGD 
               
               
                   
               
               
                  901 
                 KLNVSIRPSH KAFHLPVDAD KTPLIMIAAG SGLAPFRGFV QERAAQIAAG RSLAPAMLFY 
               
               
                   
               
               
                  961 
                 GCRHPEQDDL YRDEFDKWES IGAVSVRRAF SRCPESQETK GCKYVGDRLW EDREEVTGLW 
               
               
                   
               
               
                 1021 
                 DRGAKVYVCG SREVGESVKK VVVRIALERQ KMIVEAREKG ELDSLPEGIV EGLKLKGLTV 
               
               
                   
               
               
                 1081 
                 EDVEVSEERA LKWFEGIRNE RYATDVFD 
               
               
                   
               
            
           
           
               
               
            
               
                 CYP97C 
                   
               
               
                 
                   Oryza sativa 
                 
               
               
                 GenBank Accession No. ABB47954 
               
               
                 &gt;gi|78708979|gb|ABB47954.1| Cytochrome P450 family protein, expressed  
               
               
                 [ Oryza   sativa  Japonica Group] 
               
               
                 SEQ ID NO: 42 
                   
               
            
           
           
               
               
               
            
               
                   1 
                 MAAAAAAAVP CVPFLCPPPP PLVSPRLRRG HVRLRLRPPR SSGGGGGGGA GGDEPPITTS 
                   
               
               
                   
               
               
                  61 
                 WVSPDWLTAL SRSVATRLGG GDDSGIPVAS AKLDDVRDLL GGALFLPLFK WFREEGPVYR 
               
               
                   
               
               
                 121 
                 LAAGPRDLVV VSDPAVARHV LRGYGSRYEK GLVAEVSEFL FGSGFAIAEG ALWTVRRRSV 
               
               
                   
               
               
                 181 
                 VPSLHKRFLS VMVDRVFCKC AERLVEKLET SALSGKPVNM EARFSQMTLD VIGLSLFNYN 
               
               
                   
               
               
                 241 
                 FDSLTSDSPV IDAVYTALKE AELRSTDLLP YWKIDLLCKI VPRQIKAEKA VNIIRNTVED 
               
               
                   
               
               
                 301 
                 LITKCKKIVD AENEQIEGEE YVNEADPSIL RFLLASREEV TSVQLRDDLL SMLVAGHETT 
               
               
                   
               
               
                 361 
                 GSVLTWTIYL LSKDPAALRR AQAEVDRVLQ GRLPRYEDLK ELKYLMRCIN ESMRLYPHPP 
               
               
                   
               
               
                 421 
                 VLIRRAIVDD VLPGNYKIKA GQDIMISVYN IHRSPEVWDR ADDFIPERFD LEGPVPNETN 
               
               
                   
               
               
                 481 
                 TEYRFIPFSG GPRKCVGDQF ALLEAIVALA VVLQKMDIEL VPDQKINMTT GATIHTTNGL 
               
               
                   
               
               
                 541 
                 YMNVSLRKVD REPDFALSGS R 
               
               
                   
               
            
           
           
               
               
            
               
                 Chimeric heme enzyme C2G9 
                   
               
               
                 SEQ ID NO: 43 
                   
               
               
                 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGAL 
                   
               
               
                   
               
               
                 EKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLIQKWARLNPNEAVDVPGDMTRLTLDT 
               
               
                   
               
               
                 IGLCGENYRENSYYRETPHPFINSMVRALDEAMHQMQRLDVQDKLMVRTKRQFRYDIQTMESLVDRMIAERKANPDE 
               
               
                   
               
               
                 NIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFALYELVKNPHVLQKAAEEAARVLVDPVPSY 
               
               
                   
               
               
                 KQVKQLKYVGMVLNEALRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDP 
               
               
                   
               
               
                 SSIPHHAYKPFGNGQRACIGMQFALHEATLVLGMILKYFTLIDHENYELDIKQTLTLKPGDFHISVQSRHQEAIHAD 
               
               
                   
               
               
                 VQAAE 
               
               
                   
               
               
                 Chimeric heme enzyme X7 
               
               
                 SEQ ID NO: 44 
                   
               
               
                 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGAL 
                   
               
               
                   
               
               
                 EKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDT 
               
               
                   
               
               
                 IGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQ 
               
               
                   
               
               
                 DEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEY 
               
               
                   
               
               
                 KQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDP 
               
               
                   
               
               
                 SSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQ 
               
               
                   
               
               
                 RKEQA 
               
               
                   
               
               
                 Chimeric heme enzyme X7-12 
               
               
                 SEQ ID NO: 45 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDEERFDKSIEGALE 
                   
               
               
                   
               
               
                 KVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTI 
               
               
                   
               
               
                 GLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQD 
               
               
                   
               
               
                 EKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYK 
               
               
                   
               
               
                 QIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDPS 
               
               
                   
               
               
                 SIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQR 
               
               
                   
               
               
                 KEQA 
               
               
                   
               
               
                 Chimeric heme enzyme C2E6 
               
               
                 SEQ ID NO: 46 
                   
               
               
                 MTIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGRVTRYLSSQRLIKEACDESRFDKNLSQALK 
                   
               
               
                   
               
               
                 FVRDFAGDGLFTSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTI 
               
               
                   
               
               
                 GLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDEN 
               
               
                   
               
               
                 IKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEYK 
               
               
                   
               
               
                 QIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPS 
               
               
                   
               
               
                 AIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPS 
               
               
                   
               
               
                 PST 
               
               
                   
               
               
                 Chimeric heme enzyme X7-9 
               
               
                 SEQ ID NO: 47 
                   
               
               
                 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLVAEVCDEERFDKSIEGAL 
                   
               
               
                   
               
               
                 EKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDT 
               
               
                   
               
               
                 IGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDSIIAERRANGDQ 
               
               
                   
               
               
                 DEKDLLARMLNVEDPETGEKLDDENIRFQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEY 
               
               
                   
               
               
                 KQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDP 
               
               
                   
               
               
                 SSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQ 
               
               
                   
               
               
                 RKEQA 
               
               
                   
               
               
                 Chimeric heme enzyme C2B12 
               
               
                 SEQ ID NO: 48 
                   
               
               
                 MKQASAIPQPKTYGPLKNLPHLEKEQLSQSLWRIADELGPIFREDFPGVSSVFVSGHNLVAEVCDEERFDKSIEGAL 
                   
               
               
                   
               
               
                 EKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDT 
               
               
                   
               
               
                 IGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDE 
               
               
                   
               
               
                 NIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFATYFLLKHPDKLKKAYEEVDRVLTDAAPTY 
               
               
                   
               
               
                 KQVLELTYIRMILNESLRLWPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDP 
               
               
                   
               
               
                 SSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQ 
               
               
                   
               
               
                 RKEQA 
               
               
                   
               
               
                 Chimeric heme enzyme TSP234 
               
               
                 SEQ ID NO: 49 
                   
               
               
                 MKETSPIPQPKTFGPLGNLPLIDKDKPTLSLIKLAEEQGPIFQIHTPAGTTIVVSGHELVKEVCDEERFDKSIEGAL 
                   
               
               
                   
               
               
                 EKVRAFSGDGLATSWTHEPNWRKAHNILMPTESQRAMKDYHEKMVDIATQLIQKWSRLNPNEEIDVADDMTRLTLDT 
               
               
                   
               
               
                 IGLCGFNYRFNSFYRDQPHPFITSMVRALDEAMNKLQRANPDDPAYDENKRQFQEDIKVMNDLVDRMIAERKANPDE 
               
               
                   
               
               
                 NIKDLLSLMLYAKDPVTGETLDDENIRYQIITFLIAGHETTSGLLSFAIYCLLTHPEKLKKAQEEADRVLTDDTPEY 
               
               
                   
               
               
                 KQIQQLKYIRMVLNETLRLYPTAPAFSLYAKEDTVLGGEYPISKGQPVTVLIPKLHRDQNAWGPDAEDFRPERFEDP 
               
               
                   
               
               
                 SSIPHHAYKPFGNGQRACIGMQFALQEATMVLGLVLKHFELINHTGYELKIKEALTIKPDDFKITVKPRKTAAINVQ 
               
               
                   
               
               
                 RKEQA