VARIANT POLYPEPTIDES CAPABLE OF AMINATING ALIPHATIC ALPHA KETO ACIDS

Abstract

Disclosed are, among other things, variant polypeptides, nucleic acids encoding the polypeptides, production of the variant polypeptides, and use of the variant polypeptides in various applications, such as screening and synthetic methods. For example, the variant polypeptides, or enzymatically-active fragments thereof, are useful for converting aliphatic keto acids to aliphatic alpha amino acids.

Description

RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/059,538, filed Oct. 3, 2014.

BACKGROUND

[0002] Synthesis of (S)-2-aminonon-8-enoic acid has been reported in the literature. Faucher, et al., reported a six step synthetic sequence for (S)-2-aminonon-8-enoic acid, which involves catalytic hydrogenation of an enamine substrate utilizing a DUPHOS ligand system as the key step for introduction of .alpha.-amino acid chirality (Org. Lett. 2004, 6, 2901-2904). Subsequently, Wang, et al., reported an enzymatic approach for the preparation of (S)-2-aminonon-8-enoic acid using acylase for the selective kinetic hydrolysis of a racemic acetamide substrate, with a theoretical step yield of 50%, in a six-step sequence (Org. Process Res. Dev. 2007, 11, 60-63). In 2008, an alternate approach involving a whole-cell catalytic system was disclosed for preparation of enantiomerically enriched (S)-2-aminonon-8-enoic acid from the corresponding hydantoin substrate (WO 2008/067981 A2). Subsequently, a different approach was reported (WO 2010/050516 A1; WO 2008/067981 A2) for (S)-2-aminonon-8-enoic acid, which was also based on selective kinetic hydrolysis of a racemic succinyl amide substrate using an L-succinylase enzyme (amidase), with a theoretical 50% step yield.

[0003] Previously disclosed methods are neither efficient nor best suited for the large-scale preparation of (S)-2-aminonon-8-enoic acid, as some of them involve multiple steps, with individual steps within a sequence possessing the limitation of a maximum 50% theoretical step yield. Thus, there is a need in the art for an improved process for preparing (S)-2-aminonon-8-enoic acid.

SUMMARY

[0004] The disclosure provides, among other things, polypeptides and enzymatically-active fragments thereof capable of aminating an aliphatic keto acid (e.g., aliphatic 2-keto acids). The enzymatic activity of the polypeptides and fragments exhibits a high level of enantioselectivity for the (S)-enantiomer form of aliphatic amino acids so aminated. The polypeptides and fragments are useful for; e.g., converting 2-oxonon-8-enoic acid, in the presence of an ammonia source, to 2-aminonon-8-enoic acid (LCAA).

[0005] Accordingly, in one aspect, the disclosure features a polypeptide comprising the amino acid sequence depicted in SEQ ID NO:2 or 13-18, wherein X is not leucine.

[0006] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence that is at least 90% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, wherein X is not leucine.

[0007] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence that is at least 90% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, wherein X is not leucine.

[0008] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions, deletions, or insertions relative to SEQ ID NO:2; wherein the amino acid sequence comprises the amino acid X at position 42; and wherein X is not leucine.

[0009] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions, deletions, or insertions relative to SEQ ID NO:13, wherein the amino acid sequence comprises the amino acid X at position 43, and wherein X is not leucine.

[0010] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions, deletions, or insertions relative to SEQ ID NO:14, wherein the amino acid sequence comprises the amino acid X at position 40, and wherein X is not leucine.

[0011] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions; deletions, or insertions relative to SEQ ID NO:15, wherein the amino acid sequence comprises the amino acid X at position 40, and wherein X is not leucine.

[0012] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions, deletions, or insertions relative to SEQ ID NO:16, wherein the amino acid sequence comprises the amino acid X at position 40, and wherein X is not leucine.

[0013] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions, deletions, or insertions relative to SEQ ID NO:17, wherein the amino acid sequence comprises the amino acid X at position 40, and wherein X is not leucine.

[0014] In another aspect, the disclosure features a polypeptide comprising an amino acid sequence having at least two amino acid substitutions; deletions, or insertions relative to SEQ ID NO:18, wherein the amino acid sequence comprises the amino acid X at position 40, and wherein X is not leucine.

[0015] In yet another aspect, the disclosure features a polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 4, 5, 6, or 20.

[0016] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:2, inclusive of the amino acid at position 42, wherein X is not leucine.

[0017] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:13, inclusive of the amino acid at position 43, wherein X is not leucine.

[0018] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:14, inclusive of the amino acid at position 40, wherein X is not leucine.

[0019] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:15, inclusive of the amino acid at position 40, wherein X is not leucine.

[0020] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:16, inclusive of the amino acid at position 40, wherein X is not leucine.

[0021] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:17, inclusive of the amino acid at position 40, wherein X is not leucine.

[0022] In yet another aspect, the disclosure features a polypeptide comprising at least ten (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:18, inclusive of the amino acid at position 40, wherein X is not leucine.

[0023] In some embodiments, any of the polypeptides described herein comprise the amino acid sequence: GPAXGG (SEQ ID NO:3), wherein X is not leucine.

[0024] In some embodiments, any of the polypeptides described herein comprise at least 50 consecutive amino acids of SEQ ID NO:2. In some embodiments, any of the polypeptides described herein comprise at least 100 consecutive amino acids of SEQ ID NO:2.

[0025] In some embodiments, any of the polypeptides described herein have an enzymatic activity that converts 2-oxonon-8-enoic acid to (S)-2-aminonon-8-enoic acid (LCAA). For example, a polypeptide described herein can, in the presence of an ammonia source, convert 2-oxonon-8-enoic acid to (S)-2-aminonon-8-enoic acid (LCAA), under the assay conditions described herein and exemplified in the working examples. In some embodiments, a polypeptide described herein has an enhanced enzymatic activity to convert 2-oxonon-8-enoic acid to (S)-2-aminonon-8-enoic acid (LCAA), as compared to wild-type, full-length B. cereus LDH, e.g., SEQ ID NO: 1.

[0026] In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or any one of 13-18, X is isoleucine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or any one of 13-18, X is valine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or 13-18, X is glycine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or any one of 13-18, X is alanine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or any one of 13-18, X is serine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or 13-18, X is threonine. In some embodiments of any of the polypeptides described herein, e.g., SEQ ID NO:2, 3, or 13-18, X can be, e.g., isoleucine, valine, glycine, alanine, serine, or threonine.

[0027] In some embodiments, the polypeptides described herein can be isolated polypeptides.

[0028] In yet another aspect, the disclosure features a nucleic acid encoding any one or more of the polypeptides described herein. Also featured are expression vectors (e.g., prokaryotic or eukaryotic) expression vectors comprising the nucleic acid. In another aspect, the disclosure features a cell, plurality of cells, or culture of cells, comprising the nucleic acid or expression vector. In another aspect, the disclosure features a method for producing a polypeptide, such as any of the polypeptides described herein. The method includes culturing the aforementioned cell, plurality of cells, or culture of cells comprising the expression vector under conditions suitable for protein expression to thereby produce a polypeptide. The method can, optionally, further include isolating the polypeptide from the cell (plurality of cells or cell culture) or from media in which the cell or cells is/are cultured.

[0029] In yet another aspect, the disclosure features a kit comprising any one of the polypeptides described herein. In some embodiments, the kit includes instructions for aminating an aliphatic keto acid. In some embodiments, the kit includes an aliphatic keto acid, one or more reaction buffers, an ammonia source, a glucose dehydrogenase, glucose, nicotinamide adenine dinucleotide (NAD; e.g., a reduced form of NAD), or combinations of any of the foregoing.

[0030] "Polypeptide," "peptide," and "protein" are used interchangeably and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0032] Other features and advantages of the present disclosure, e.g., methods for reductive amination of an aliphatic keto acid, will be apparent from the following description, the examples, and from the claims.

BRIEF DESCRIPTION OF THE SEQUENCES

[0033] SEQ ID NO:1 is an exemplary amino acid sequence for wild-type B. cereus leucine dehydrogenase.

[0034] SEQ ID NO:2 is an exemplary amino acid sequence for a variant B. cereus leucine dehydrogenase (substitution X at position 42).

[0035] SEQ ID NO:3 is a six amino acid conserved region from bacterial leucine dehydrogenase polypeptides.

[0036] SEQ ID NO:4 depicts an exemplary amino acid sequence for the variant B. cereus LDH-L42I polypeptide.

[0037] SEQ ID NO:5 depicts an exemplary amino acid sequence for the variant B. cereus LDH-L42V polypeptide.

[0038] SEQ ID NO:6 depicts an exemplary amino acid sequence for the variant B. cereus LDH-L42G polypeptide.

[0039] SEQ ID NO:7 is an exemplary amino acid sequence for wild-type Chlamydia pneumoniae leucine dehydrogenase.

[0040] SEQ ID NO:8 is an exemplary amino acid sequence for wild-type Thermoactinomyces intermedius leucine dehydrogenase.

[0041] SEQ ID NO:9 is an exemplary amino acid sequence for wild-type Bacillus subtilis leucine dehydrogenase.

[0042] SEQ ID NO:10 is an exemplary amino acid sequence for wild-type Bacillus lichenformis leucine dehydrogenase.

[0043] SEQ ID NO:11 is an exemplary amino acid sequence for wild-type Geobacilllus stearothermophilus leucine dehydrogenase.

[0044] SEQ ID NO:12 is an exemplary amino acid sequence for wild-type Bacillus sphaericus leucine dehydrogenase.

[0045] SEQ ID NO:13 is an exemplary amino acid sequence for a variant Chlamydia pneumoniae leucine dehydrogenase (substitution X at position 43).

[0046] SEQ ID NO:14 is an exemplary amino acid sequence for a variant Thermoactinomyces intermedius leucine dehydrogenase (substitution X at position 40).

[0047] SEQ ID NO:15 is an exemplary amino acid sequence for a variant Bacillus subtilis leucine dehydrogenase (substitution X at position 40).

[0048] SEQ ID NO:16 is an exemplary amino acid sequence for a variant Bacillus lichenformis leucine dehydrogenase (substitution X at position 40).

[0049] SEQ ID NO:17 is an exemplary amino acid sequence for a variant Geobacillus stearothermophilus leucine dehydrogenase (substitution X at position 40).

[0050] SEQ ID NO:18 is an exemplary amino acid sequence for a variant Bacillus sphaericus leucine dehydrogenase (substitution X at position 40).

[0051] SEQ ID NO:19 is a three amino acid conserved region from bacterial leucine dehydrogenase polypeptides.

[0052] SEQ ID NO:20 depicts an exemplary acid sequence for the variant B. cereus LDH-L42A polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 depicts the alignment of the amino acid sequences of leucine dehydrogenases from Bacillus cereus (BC) (SEQ ID NO:1), Chlamydia pneumoniae (CP) (SEQ ID NO:7), Thermoactinomyces intermedius (TI) (SEQ ID NO:8), Bacillus subtilis (BS) (SEQ ID NO:9), Bacillus licheniformis (BL) (SEQ ID NO: 10), Geobacillus stearothermophilus (GS) (SEQ ID NO:11), and Bacillus sphaericus (BSph) (SEQ ID NO:12). Sequence identity is indicated by "*". Sequence conservation (e.g., conservative substitutions) between species is indicated by ":". The alignment was performed using the ClustalW2.TM. software provided by the European Bioinformatics Institute (EBI) of the European Molecular Biology Laboratory (EMBL).

[0054] FIG. 2 depicts a reaction scheme for converting 2-oxonon-8-enoic acid, in the presence of an ammonia source, to 2-aminonon-8-enoic acid (LCAA). Also depicted, in conjunction with the amination reaction, are glucose dehydrogenase (GDH) and glucose, which are used to regenerate a catalytic amount of the NADH cofactor.

[0055] FIG. 3 is a line graph depicting the rate of conversion of 2-oxonon-8-enoic acid (substrate) to (S)-LCAA (product) using wild-type, full-length Bacillus cereus LDH. The Y-axis represents the percent conversion of substrate to product. The X-axis represents time in minutes.

[0056] FIG. 4 is a chromatograph depicting the percentage of (S)-enantiomer of LCAA produced. by the LDH-driven enzymatic reaction. Analyses of a racemic standard having both (S) and (R) forms, as well as chemically synthesized (S)-LCAA, were also performed (and shown) for reference.

[0057] FIG. 5 is a line graph depicting the relative reaction rates of wild-type LDH and L32I, L42V, and L42G LDH variants for converting 2-oxonon-8-enoic acid (substrate) to (S)-LCAA. The Y-axis represents the relative percent conversion of substrate to product. The X-axis represents time.

DETAILED DESCRIPTION

[0058] The disclosure relates to, among other things, polypeptides (e.g., variant polypeptides and enzymatically-active fragments thereof), nucleic acids encoding the polypeptides, production of the polypeptides, and use of the polypeptides in various applications, such as screening and synthetic methods. For example, the variant polypeptides, or enzymatically-active fragments thereof, are useful for converting aliphatic keto acids to aliphatic alpha amino acids. While in no way intended to be limiting, exemplary variant polypeptides, fragments, and methods for making and using any of the foregoing are elaborated on below.

Polypeptides

[0059] The polypeptides described herein include variants of leucine dehydrogenase (LDH) and enzymatically-active fragments of such variants. In some embodiments, the polypeptide is a variant of a LDH expressed by Bacillus cereus, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type polypeptide from Bacillus cereus is as follows:

TABLE-US-00001 (UniProt ID No. P0A392) (SEQ ID NO: 1) MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGGTRMWTY DSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRA LGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSP VTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAK LIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETI PQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVTNAGGVINVADEL YGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKN SRSTYLRNGHDIISRR.

[0060] In some embodiments, the polypeptide is a variant of a LDH expressed by Chlamydia pneumoniae, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Chlamydia pneumoniae is as follows:

TABLE-US-00002 (UniProt ID No. Q9Z6Y7) (SEQ ID NO: 7) MKYSLNFKEIKIDDYERVIEVTCSKVRLHAIIAIHQTAVGPALGGVRASL YSSFEDACTDALRLARGMTYKAIISNTGTGGGKSVIILPQDAPSLTEDML RAFGQAVNALEGTYICAEDLGVSINDISIVAEETPYVCGIADVSGDPSIY TAHGGFLCIKETAKYLWGSSSLRGKKIAIQGIGSVGRRLLQSLFFEGAEL YVADVLERAVQDAARLYGATIVPTEEIHALECDIFSPCARGNVIRKDNLA DLNCKAIVGVANNQLEDSSAGMMLHERGILYGPDYLVNAGGLLNVAAAIE GRVYAPKEVLLKVEELPIVLSKLYNQSKTTGKDLVALSDSFVEDKLLAYT S.

[0061] In some embodiments, the polypeptide is a variant of a LDH expressed by Thermoactinomyces intermedius, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Thermoactinomyces intermedius is as follows:

TABLE-US-00003 (UniProt ID No. Q60030) (SEQ ID NO: 8) MKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPALGGMRMWTYAS EEEAIEDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRALG RFIQGLNGRYITAEDVGTTVEDMDIIHEETRYVTGVSPAFGSSGNPSPVT AYGVYRGMKAAAKEAFGDDSLEGKVVAVQGVGHVAYELCKHLHNEGAKLI VTDINKENADRAVQEFGAEFVHPDKIYDVECDIFAPCALGAIINDETIER LKCKVVAGSANNQLKEERHGKMLEEKGIVYAPDYVINAGGVINVADELLG YNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADRMAEERIEMMRKTR STFLQDQRNLINFNNK.

[0062] In some embodiments, the polypeptide is a variant of a LDH expressed by Bacillus subtilis, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Bacillus subtilis is as follows:

TABLE-US-00004 (UniProt ID No. P54531) (SEQ ID NO: 9) MELFKYMEKYDYEQLVFCQDEQSGLKAIIAIHDTTLGPALGGTRMWTYEN EEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFG RYIQGLNGRYITAEDVGTTVEDMDIIHDETDYVTGISPAFGSSGNPSPVT AYGVYRGMKAAAKAAFGTDSLEGKTIAVQGVGNVAYNLCRHLHEEGANLI VTDINKQSVQRAVEDFGARAVDPDDIYSQDCDIYAPCALGATINDDTIKQ LKAKVIAGAANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYG YNAERALKKVEGIYGNIERVLEISQRDGIPAYLAADRLAEERIERMRRSR SQFLQNGHSVLSRR.

[0063] In some embodiments, the polypeptide is a variant of a LDH expressed by Bacillus lichenformis, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Bacillus licheniformis is as follows:

TABLE-US-00005 (UniProt ID No. Q65HK5) (SEQ ID NO: 10) MELFRYMEQYDYEQLVFCQDKQSGLKAIIAIHDTTLGPALGGTRMWTYES EEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFG RYIQGLNGRYITAEDVGTTVEDMDIIHDETDFVTGISPAFGSSGNPSPVT AYGVYKGMKAAAKAAFGTDSLEGKTVAVQGVGNVAYNLCRHLHEEGAKLI VTDINKEAVERAVAEFGARAVDPDDIYSQECDIYAPCALGATINDDTIPQ LKAKVIAGAANNQLKETRHGDQIHDMGIVYAPDYVINAGGVINVADELYG YNSERALKKVEGIYGNIERVLEISKRDRIPTYLAADRLAEERIERMRQSR SQFLQNGHHILSRR.

[0064] In some embodiments, the polypeptide is a valiant of a LDH expressed by Geobacillus stearothermophilus, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Geobacillus stearothermophilus is as follows:

TABLE-US-00006 (UniProt ID No. P13154) (SEQ ID NO: 11) MELFKYMETYDYEQVLFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYNS EEEALEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRAFG RFIQGLNGRYITAEDVGTTVADMDIIYQETDYVTGISPEFGSSGNPSPAT AYGVYRGMKAAAKEAFGSDSLEGKVVAVQGVGNVAYHLCRHLHEEGAKLI VTDINKEVVARAVEEFGAKAVDPNDIYGVECDIFAPCALGGIINDQTIPQ LKAKVIAGSADNQLKEPRHGDIIHEMGIVYAPDYVINAGGVINVADELYG YNRERAMKKIEQIYDNIEKVFAIAKRDNIPTYVAADRMAEERIETMRKAR SPFLQNGHHILSRRRAR.

[0065] In some embodiments, the polypeptide is a variant of a LDH expressed by Bacillus sphaericus, or an enzymatically-active fragment of the variant. An exemplary amino acid sequence for the full-length, wild-type LDH polypeptide from Bacillus sphaericus is as follows:

TABLE-US-00007 (UniProt ID No. Q76GS2) (SEQ ID NO: 12) MEIFKYMEKYDYEQLVFCQDEASGLKAIIAIHDTTLGPALGGARMWTYAT EENAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPFKDKNEEMFRALG RFIQGLNGRYITAEDVGTTVTDMDLIHEETNYVTGISPAFGSSGNPSPVT AYGVYRGMKAAAKEAFGTDMLEGRTISVQGLGNVAYKLCEYLHNEGAKLV VTDINQAAIDRVVNDFGATAVAPDEIYSQEVDIFSPCALGAILNDETIPQ LKAKVIAGSANNQLQDSRHGDYLHELGIVYAPDYVINAGGVINVADELYG YNRERALKRVDGIYDSIEKIFEISKRDSIPTYVAANRLAEERIARVAKSR SQFLKNEKNILNGR.

[0066] The variant polypeptides described herein comprise one or more amino acid substitutions, insertions, or deletions, relative to the wild-type LDH polypeptides from which they were derived. In some embodiments, a variant polypeptide comprises at least two (e.g., at least three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100) amino acid substitutions, deletions, or insertions, relative to the wild-type, full-length LDH polypeptide from which it was derived. In some embodiments, a variant polypeptide comprises no more than 150 (e.g., no more than 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid substitutions, deletions, or insertions, relative to the wild-type, full-length LDH polypeptide from which it was derived. In some embodiments, a variant polypeptide described herein, or a fragment thereof, includes an amino acid substitution at amino acid position 42 relative to SEQ ID NO:1, e.g., a substitution of leucine at position 42 with another amino acid. The amino acid at position 42, leucine, relative to SEQ ID NO:1 is one of several amino acids (GPAXGG (SEQ ID NO:3)) highly conserved among bacterial leucine dehydrogenase polypeptides (FIG. 1). However, the exact position of these amino acid residues in a given polypeptide varies from species to species and with truncations or extension of the naturally-occurring sequence. One of skill in the art would therefore appreciate that references herein to a variant polypeptide (or a fragment thereof) comprising an amino acid substitution at position 42 relative to SEQ ID NO:1, include, e.g., an amino acid substitution at position 43 of SEQ ID NO:7; an amino acid substitution at position 40 of SEQ ID NO:8; an amino acid substitution at position 40 of SEQ ID NO:9; an amino acid substitution at position 40 of SEQ ID NO:10; an amino acid substitution at position 40 of SEQ ID NO:11; or an amino acid substitution at position 40 of SEQ ID NO:12, i.e., position X in SEQ ID NOs:13-18.

[0067] In some embodiments, any of the variant polypeptides or fragments described herein comprise the amino acid sequence NVA (SEQ ID NO:19), which corresponds to amino acids 295 to 297 of SEQ ID NO:1. In some embodiments, a variant polypeptide or fragment thereof comprises the amino acid sequences depicted in SEQ ID NO:3 and SEQ ID NO:19.

[0068] As used herein, the term "conservative substitution" refers to the replacement of an amino acid present in the native sequence in a given polypeptide with a naturally or non-naturally occurring amino acid having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic; the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid that is also polar or hydrophobic, and, optionally, with the same or similar steric properties as the side-chain of the replaced amino acid. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. One letter amino acid abbreviations are as follows: alanine (A); arginine (R); asparagine (N); aspartic acid (D); cysteine (C); glycine (G); glutamine (Q); glutamic acid (E); histidine (H); isoleucine (I); leucine (L); lysine (K); methionine (M); phenylalanine (F); proline (P); serine (S); threonine (T); tryptophan (W), tyrosine (Y); and valine (V).

[0069] The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted.

[0070] In some embodiments, the variant polypeptide, or fragment thereof, comprises the amino acid sequence GPAXGG (SEQ ID NO:3), wherein X is any amino acid except for leucine. In some embodiments, X is glycine. In some embodiments, X is valine. In some embodiments, X is isoleucine. In some embodiments, X is alanine. In some embodiments, X is serine. In some embodiments, X is threonine. In some embodiments, X can be, e.g., glycine, valine, isoleucine, alanine, serine, or threonine.

[0071] In some embodiments, the variant polypeptide is a variant of Bacillus cereus LDH comprising the following amino acid sequence: MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPAXGGTRMWTYDSEEAAIED ALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAED VGTTVDDMDIIHEETDFVTGISPSFGSSGNPSVTAYGVYRGMKAAAKEAFGTDNLEGK VIAVQGVGNVAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDI YAPCALGATVNDETIPQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVI NVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRST YLRNGHDIISRR (SEQ ID NO:2), wherein X is any amino acid except for leucine. In some embodiments, X is glycine. In some embodiments, X is valine. In some embodiments, X is isoleucine. In some embodiments, X is alanine. In some embodiments, X is serine. In some embodiments, X is threonine. In some embodiments, X can be, e.g., glycine, valine, isoleucine, alanine, serine, or threonine.

[0072] In some embodiments, the variant polypeptide comprises, or consists of, one of the following amino acid sequences:

TABLE-US-00008 (1) (SEQ ID NO: 4) MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPAIGGTRMWTY DSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRA LGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSP VTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAK LIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETI PQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADEL YGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKN SRSTYLRNGHDIISRR; (2) (SEQ ID NO: 5) MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPAVGGTRMWTY DSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRA LGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSP VTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAK LIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETI PQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADEL YGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKN SRSTYLRNGHDIISRR; (3) (SEQ ID NO: 6) MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPAGGGTRMWTY DSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRA LGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSP VTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAK LIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETI PQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADEL YGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKN SRSTYLRNGHDIISRR, or (4) (SEQ ID NO: 20) MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPAAGGTRMWTY DSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRA LGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSP VTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGNVAYHLCKHLHAEGAK LIVTDINKEAVQRAVEEFGASAVEPNEIYGWCDIYAPCALGATVNDETIP QLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADELY GYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNS RSTYLRNGHDIISRR.

[0073] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:2, inclusive of the amino acid at position 42, wherein X is not leucine.

[0074] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:13, inclusive of the amino acid at position 43, wherein X is not leucine. The amino acid sequence of SEQ ID NO:13 is as follows:

TABLE-US-00009 MKYSLNFKEIKIDDYERVIEVTCSKVRLHAIIAIHQTAVGPAXGGVRASL YSSFEDACTDALRLARGMTYKAIISNTGTGGGKSVIILPQDAPSLTEDML RAFGQAVNALEGTYICAEDLGVSINDISIVAEETPYVCGIADVSGDPSIY TAHGGFLCIKETAKYLWGSSSLRGKKIAIQGIGSVGRRLLQSLFFEGAEL YVADVLERAVQDAARLYGATIVPTEEIHALECDIFSPCARGNVIRKDNLA DLNCKAIVGVANNQLEDSSAGMMLHERGILYGPDYLVNAGGLLNVAAAIE GRVYAPKEVLLKVEELPIVLSKLYNQSKTTGKDLVALSDSFVEDKLLAYT S.

[0075] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:14, inclusive of the amino acid at position 40, wherein X is not leucine. The amino acid sequence of SEQ ID NO:14 is as follows:

TABLE-US-00010 MKIFDYMEKYDYEQLVMCQDKESGLKAIICIHVTTLGPAXGGMRMWTYAS EEEAIEDALRLGRGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRALG RFIQGLNGRYITAEDVGTTVEDMDIIHEETRYVTGVSPAFGSSGNPSPVT AYGVYRGMKAAAKEAFGDDSLEGKVVAVQGVGHVAYELCKHLHNEGAKLI VTDINKENADRAVQEFGAEFVHPDKIYDVECDIFAPCALGAIINDETIER LKCKVVAGSANNQLKEERHGKMLEEKGIVYAPDYVINAGGVINVADELLG YNRERAMKKVEGIYDKILKVFEIAKRDGIPSYLAADRMAEERIEMMRKTR STFLQDQRNLINFNNK.

[0076] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:15, inclusive of the amino acid at position 40, wherein X is not leucine. The amino acid sequence of SEQ ID NO:15 is as follows:

TABLE-US-00011 MELFKYMEKYDYEQLVFCQDEQSGLKAIIAIHDTTLGPAXGGTRMWTYEN EEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFG RYIQGLNGRYITAEDVGTTVEDMDIIHDETDYVTGISPAFGSSGNPSPVT AYGVYRGMKAAAKAAFGTDSLEGKTIAVQGVGNVAYNLCRHLHEEGANLI VTDINKQSVQRAVEDFGARAVDPDDIYSQDCDIYAPCALGATINDDTIKQ LKAKVIAGAANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYG YNAERALKKVEGIYGNIERVLEISQRDGIPAYLAADRLAEERIERMRRSR SQFLQNGHSVLSRR.

[0077] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:16, inclusive of the amino acid at position 40, wherein X is not leucine. The amino acid sequence of SEQ ID NO:16 is as follows:

TABLE-US-00012 MELFRYMEQYDYEQLVFCQDKQSGLKAIIAIHDTTLGPAXGGTRMWTYES EEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFG RYIQGLNGRYITAEDVGTTVEDMDIIHDETDFVTGISPAFGSSGNPSPVT AYGVYKGMKAAAKAAFGTDSLEGKTVAVQGVGNVAYNLCRHLHEEGAKLI VTDINKEAVERAVAEFGARAVDPDDIYSQECDIYAPCALGATINDDTIPQ LKAKVIAGAANNQLKETRHGDQIHDMGIVYAPDYVINAGGVINVADELYG YNSERALKKVEGIYGNIERVLEISKRDRIPTYLAADRLAEERIERMRQSR SQFLQNGHHILSRR.

[0078] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least ten (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:17, inclusive of the amino acid at position 40, wherein X is not leucine. The amino acid sequence of SEQ ID NO:17 is as follows:

TABLE-US-00013 MELFKYMETYDYEQVLFCQDKESGLKAIIAIHDTTLGPAXGGTRMWMYNS EEEALEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEAMFRAFG RFIQGLNGRYITAEDVGTTVADMDIIYQETDYVTGISPEFGSSGNPSPAT AYGVYRGMKAAAKEAFGSDSLEGKVVAVQGVGNVAYHLCRHLHEEGAKLI VTDINKEVVARAVEEFGAKAVDPNDIYGVECDIFAPCALGGIINDQTIPQ LKAKVIAGSADNQLKEPRHGDIIHEMGIVYAPDYVINAGGVINVADELYG YNRERAMKKIEQIYDNIEKVFAIAKRDNIPTYVAADRMAEERIETMRKAR SPFLQNGHHILSRRRAR.

[0079] In some embodiments, a variant polypeptide described herein, or a fragment thereof, comprises at least 10 (e.g., at least 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 or more) consecutive amino acids of SEQ ID NO:18, inclusive of the amino acid at position 40, wherein X is not leucine. The amino acid sequence of SEQ ID NO:18 is as follows:

TABLE-US-00014 MEIFKYMEKYDYEQLVFCQDEASGLKAIIAIHDTTLGPAXGGARMWTYAT EENAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPFKDKNEEMFRALG RFIQGLNGRYITAEDVGTTVTDMDLIHEETNYVTGISPAFGSSGNPSPVT AYGVYRGMKAAAKEAFGTDMLEGRTISVQGLGNVAYKLCEYLHNEGAKLV VTDINQAAIDRVVNDFGATAVAPDEIYSQEVDIFSPCALGAILNDETIPQ LKAKVIAGSANNQLQDSRHGDYLHELGIVYAPDYVINAGGVINVADELYG YNRERALKRVDGIYDSIEKIFEISKRDSIPTYVAANRLAEERIARVAKSR SQFLKNEKNILNGR.

[0080] In some embodiments of any of the variants described herein, X is glycine, isoleucine, alanine, or valine. In some embodiments, X is serine. In some embodiments; X is threonine. In some embodiments, X can be, e.g., glycine, valine, isoleucine, alanine, serine, and threonine.

[0081] In some embodiments, a variant polypeptide described herein, or a fragment thereof, has an amino acid sequence that is at least 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99)% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, with the proviso that the variant polypeptide or fragment thereof comprises the amino acid sequence at position X, and X is not leucine. In some embodiments, the variant polypeptide or fragment thereof comprises the amino acid sequence depicted in SEQ ID NO:3, wherein X is not leucine.

[0082] In some embodiments, a variant polypeptide described herein, or a fragment thereof, has an amino acid sequence that is at least 80 (e.g., at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99)% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ. ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, with the proviso that the variant polypeptide or fragment thereof comprises the amino acid sequence at position X; and X is not leucine. In some embodiments, the variant polypeptide or fragment thereof comprises the amino acid sequence depicted in SEQ ID NO:3, wherein X is not leucine.

[0083] Percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary; to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art; for instance, using publicly available computer software, such as BLAST software or ClustalW2 (above). Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0084] Leucine dehydrogenase from B. cereus exists in solution as a homo-octomer, with each subunit folding into two domains, and separated by a deep cleft. See Baker et al. (1995) Current Biol 3:693-705, which describes the crystal structure of leucine dehydrogenase from B. sphaericus (SEQ ID NO:12). The quaternary structure of the complex adopts the shape of a hollow cylinder. Leucine dehydrogenase comprises both a dehydrogenase superfamily domain (e.g., amino acids 10 to 130) and a nicotinamide adenine dinucleotide-cofactor binding domain (e.g., amino acids 150 to 350). In some embodiments, a variant polypeptide or enzymatically-active fragment described herein retains at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100)% of the ability of the corresponding full-length, wild-type LDH polypeptide from which the variant or fragment was derived to bind to a nucleotide cofactor (e.g., NAD or NADH). Methods for detecting or measuring the interaction between NAD and NAD-dependent enzymes are known in the art and described in, e.g., Kovar and Klukanova (1984) Biochim Biophys Acta 788(1):98-109, and Lesk (1995) Curr Opin Struct Biol 5(6): 775-783.

[0085] As described above, the variant polypeptides described herein, as well as enzymatically-active fragments thereof, possess an enzymatic activity capable of reductive amination of an aliphatic keto acid (e.g., aliphatic 2-keto acids). For example, such polypeptides can convert 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA, e.g., (S)-LCAA. In some embodiments, a variant polypeptide, or enzymatically-active fragment thereof, retains at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100)% of the ability of the corresponding full-length, wild-type LDH polypeptide from which the variant or fragment was derived to convert 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA. In some embodiments, a variant polypeptide, or enzymatically-active fragment thereof, retains at least 5 (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100)% of the ability of full-length, wild-type Bacillus cereus LDH octomer complex to convert 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA, e.g., under the assay conditions described and exemplified in the working examples.

[0086] In some embodiments, a variant polypeptide, or enzymatically-active fragment thereof, possesses enhanced ability to convert 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA, relative to the activity of full-length, wild-type Bacillus cereus LDH. For example, the variant polypeptide or enzymatically-active fragment thereof can have at least a 5 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100)% greater activity (e.g., reaction rate) than hill-length, wild-type Bacillus cereus LDH to convert 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA. In some embodiments, the activity (e.g., the reaction rate) of the variant polypeptide or enzymatically-active fragment thereof is at least 1.5 (e.g., at least 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 500, or even 1000) times greater than that of full-length, wild-type Bacillus cereus LDH, e.g., under the conditions described and exemplified in the working examples. Exemplary variant polypeptides exhibiting enhanced activity relative to full-length, wild-type B. cereus LDH include the L42I, L42V, L42G, and L32A variant polypeptides having amino acid sequences: SEQ ID NOs:4, 5, 6, and 20, respectively (see Example 2).

Recombinant Protein Expression and Purification

[0087] The variant polypeptides (or fragments) described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. (See, e.g., Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). For example, a nucleic acid encoding the variant polypeptide or fragment can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. In addition, the choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and may be easily optimized as needed.

[0088] The variant polypeptides or fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the proteins or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, proteins expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001), supra).

[0089] A number of expression systems for use in prokaryotic host cells have been described, e.g., Terpe et al. (2006) Appl Microbiol Biotechnol 72:211-222. Commonly used expression systems for bacterial host cells (e.g., E. coli) include: the lac promoter, trc and tac promoter systems, T7 systems, phage promoter Pt., tetA promoter/operator, araBAD promoter, and rhaPBAD promoter systems. See, e.g., Polisky et al. (1976) Proc Natl Acad Sci USA 73:3900-3904; De Boer et al. (1983) Proc Natl Acad Sci USA 80:21-25; Brosius et al. (1985) J Biol Chem 260:3539-3541; Amann et al. (1983) Gene 25:167-178; and Quick and Wright (2002) Proc Natl Acad Sci USA 99:8597-8601. Another widely-used bacterial expression system is the pET vector expression system. Studier et al. (1986) J Mol Biol 189:113-130; Dietrich et al. (1991) Eur J Biochem 201:399-407; Lathrop et al. (1992) Protein Expr Purif 3:512-517; and Aukhil et al. (1993) J Biol Chem 268:2542-2553.

[0090] Several possible vector systems are available for the expression of recombinant polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol Appl Genet 1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979) Cell 16:77). A second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan (1981) Nature 293:79).

[0091] The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaPO.sub.4 precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection.

[0092] Appropriate host cells for the expression of recombinant proteins include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines. In addition to E. coli, other commonly-used prokaryotic host cells include bacilli strains B. megaterium, B. subtilis, B. brevis, and Caulohacter crescentus.

[0093] A variant polypeptide or fragment thereof described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and even in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0094] Following expression, the recombinant proteins can be isolated. The term "purified" or "isolated" as applied to any of the proteins described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryotic or eukaryotic cell expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99)%, by weight, of the total protein in a sample.

[0095] The recombinant proteins can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) "Protein Purification, 3.sup.rd edition," Springer-Verlag, New York City, N.Y. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed proteins will be necessary.

[0096] Methods for determining the yield or purity of a purified protein are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain).

[0097] Exemplary methods for producing, expressing, and isolating a LDH from E. coli are exemplified in the working examples. Additional methods for producing a recombinant LDH (e.g., recombinant LDH from B. sphaericus) are described in, e.g., Li et al. (2009) Appl Biochem Biotechnol 158:343-351.

Applications

[0098] The variant polypeptides and enzymatically-active fragments thereof are useful in a number of applications. For example, the polypeptides and fragments can be used as control enzymes in screening methods designed to identify additional variant polypeptides and fragments capable of converting 2-oxonon-8-enoic acid, in the presence of an ammonia source, to LCAA, e.g., (S)-LCAA. Such methods would include, optionally, generating one or more (e.g., a library of) test variant leucine dehydrogenase polypeptides (e.g., substitution, insertion, or deletion variants of any one of SEQ ID NOs:1, 2, or 4-18). Methods for generating test variant polypeptides are described herein (supra) and exemplified in the working examples. The test variant polypeptides can be screened, e.g., using the LCAA production reaction described in Examples 1 and 2 below.

[0099] In addition, the variant polypeptides and enzymatically-active fragments thereof described herein are useful as enzyme catalysts for the conversion of 2-oxonon-8-enoic acid to (S)-LCAA. Such methods are described herein and exemplified in the working examples.

Kits

[0100] Also provided herein are kits containing one or more of the variant polypeptides or enzymatically-active fragments thereof and, optionally, instructions for carrying out a reaction to aminate an aliphatic keto acid. The variant polypeptides or fragments can be provided in solution, e.g., aqueous solution, or in lyophilized form. In the latter case, the kit can optionally include one or more buffers for reconstituting the lyophilized protein.

[0101] In some embodiments, the kits can include nicotinamide adenine dinucleotide (NAD), e.g., a reduced form of NAD and, optionally, appropriate reaction buffers. In some embodiments, the kits can include glucose. In some embodiments, the kits can further include a glucose dehydrogenase.

EXAMPLES

[0102] The following examples are intended to illustrate, not limit, the disclosure.

Example 1. Production of 2-aminonon-8-enoic Acid (LCAA) Using Leucine Dehydrogenase

[0103] E. coli cells were transformed with an expression vector encoding wild-type, B. cereus leucine dehydrogenase (LDH; having the amino acid sequence depicted in SEQ ID NO:1). LDH protein was expressed at 37.degree. C. using standard molecular biology techniques. Briefly, seed cultures of 10 mL of LB media with 50 .mu.g/mL ampicillin were inoculated with frozen cell stocks of E. coli containing the expression vector. The cultures were incubated at 37.degree. C. and 250 rpm overnight. Expression cultures were inoculated with the seed culture at a 1:200 dilution and grown at 37.degree. C. and 250 rotations per minute (rpm) until the culture reached an OD.sub.600 of 0.5, at which time they were induced with 0.5 .mu.L of 0.2 M IPTG. After induction, cultures were incubated at 37.degree. C. for 18-24 hours.

[0104] Following expression, cultures were harvested by centrifugation (14,000 rpm for 3 min) and resuspended with 1/10th the original culture volume in B-PER.TM. Protein Extraction Reagent (Thermo Scientific, Rockford, Ill.). Alternatively, cell cultures could also be lysed by resuspension in buffer (i.e. 0.1 M phosphate buffer pH 7) followed by sonication. The resulting cell lysate was clarified by centrifugation at 14,000 rpm for 3 min. The supernatant was reserved as the cell-free extract LDH enzyme solution.

[0105] To produce (S)-LCAA from 2-oxonon-8-enoic acid, a reaction was performed under the following conditions. In 200 mL total volume and a buffered pH of 9.5, the aqueous reaction mixture contained 10 mM 2-oxonon-8-enoic acid, 12 mM glucose, 1 mM NAD.sup.+, 50 mg of purified glucose dehydrogenase (GDH-105; Codexis, Redwood City, Calif.), 2 M NH.sub.4Cl/OH, and approximately 60 mL/L of purified leucine dehydrogenase cell-free extract. FIG. 2 depicts the reaction scheme for converting 2-oxonon-8-enoic acid to (S)-LCAA using leucine dehydrogenase. The mixture was incubated at 30.degree. C. and with shaking at 150 rpm for four hours. Enzyme activity was measured spectrophotometrically by monitoring the consumption of NADH in the reductive amination at 340 nm. Activity was defined as the number of micromoles of NADH consumed in 1 minute (.mu.mol min.sup.-1). Aliquots of the reaction mixture were obtained periodically during the course of the reaction and subjected to analysis by high performance liquid chromatography (HPLC). As shown in FIG. 3, greater than 99.5% conversion of the substrate to LCAA was achieved by four hours.

[0106] To determine what percentage of the LCAA reaction product of the reaction was (S)-LCAA, aliquots of the reaction mixture were analyzed using high performance liquid chromatography (HPLC). As shown in FIG. 4, greater than 99.5% of the reaction product was the desired (S)-enantiomer.

Example 2. Exemplary Variant B. cereus LDH Polypeptides

[0107] To increase the affinity of LDH for the 2-oxonon-8-enoic acid substrate and, thus, enhance the enzyme's activity for producing (S)-LCAA, amino acid substitutions were made within the region of LDH that binds to the substrate. Specifically, amino acid substitutions were introduced at position 42 of B. cereus leucine dehydrogenase (SEQ ID NO:1) using standard mutagenesis techniques. Wild-type B. cereus LDH, along with 19 variants (one for each amino acid substitution at position 42) were expressed and isolated as described in Example 1. The enzymatic activity of the variant proteins was evaluated alongside wild-type B. cereus LDH in the reaction described in Example 1. As shown in FIG. 5, three of the substitution variants (L42I, L42V, and L42G) exhibited significantly enhanced activity (a greater reaction rate), relative to wild-type B. cereus LDH. L42G and L42V exhibited an approximately 1.times.10.sup.3 increase in reaction rate, relative to wild-type B. cereus LDH. The L42A, L42T, and L42S variants also exhibited enhanced activity relative to wild-type B. cereus LDH. The L42A variant had a similar level of enzymatic activity to the L42V variant LDH enzyme. By contrast, L42D, L42K, L42Y, and L42H variants possessed reduced activity, relative to wild-type B. cereus LDH, under these reaction conditions. While the disclosure is not bound by any particular theory or mechanism of action, the substitutions that increased the enzymatic activity of the LDH enzyme are believed to have increased the depth of the substrate binding pocket in LDH and thereby increased the affinity of LDH for the 2-oxonon-8-enoic acid substrate.

[0108] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

Sequence CWU 1

1

201366PRTBacillus cereus 1Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365 2366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(42)..(42)Any amino acid except Leu 2Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Xaa Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365 36PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptideMOD_RES(4)..(4)Any amino acid except Leu 3Gly Pro Ala Xaa Gly Gly 1 5 4366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 4Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Ile Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365 5366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 5Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Val Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365 6366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 6Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Gly Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365 7351PRTChlamydia pneumoniae 7Met Lys Tyr Ser Leu Asn Phe Lys Glu Ile Lys Ile Asp Asp Tyr Glu 1 5 10 15 Arg Val Ile Glu Val Thr Cys Ser Lys Val Arg Leu His Ala Ile Ile 20 25 30 Ala Ile His Gln Thr Ala Val Gly Pro Ala Leu Gly Gly Val Arg Ala 35 40 45 Ser Leu Tyr Ser Ser Phe Glu Asp Ala Cys Thr Asp Ala Leu Arg Leu 50 55 60 Ala Arg Gly Met Thr Tyr Lys Ala Ile Ile Ser Asn Thr Gly Thr Gly 65 70 75 80 Gly Gly Lys Ser Val Ile Ile Leu Pro Gln Asp Ala Pro Ser Leu Thr 85 90 95 Glu Asp Met Leu Arg Ala Phe Gly Gln Ala Val Asn Ala Leu Glu Gly 100 105 110 Thr Tyr Ile Cys Ala Glu Asp Leu Gly Val Ser Ile Asn Asp Ile Ser 115 120 125 Ile Val Ala Glu Glu Thr Pro Tyr Val Cys Gly Ile Ala Asp Val Ser 130 135 140 Gly Asp Pro Ser Ile Tyr Thr Ala His Gly Gly Phe Leu Cys Ile Lys 145 150 155 160 Glu Thr Ala Lys Tyr Leu Trp Gly Ser Ser Ser Leu Arg Gly Lys Lys 165 170 175 Ile Ala Ile Gln Gly Ile Gly Ser Val Gly Arg Arg Leu Leu Gln Ser 180 185 190 Leu Phe Phe Glu Gly Ala Glu Leu Tyr Val Ala Asp Val Leu Glu Arg 195 200 205 Ala Val Gln Asp Ala Ala Arg Leu Tyr Gly Ala Thr Ile Val Pro Thr 210 215 220 Glu Glu Ile His Ala Leu Glu Cys Asp Ile Phe Ser Pro Cys Ala Arg 225 230 235 240 Gly Asn Val Ile Arg Lys Asp Asn Leu Ala Asp Leu Asn Cys Lys Ala 245 250 255 Ile Val Gly Val Ala Asn Asn Gln Leu Glu Asp Ser Ser Ala Gly Met 260 265 270 Met Leu His Glu Arg Gly Ile Leu Tyr Gly Pro Asp Tyr Leu Val Asn 275 280 285 Ala Gly Gly Leu Leu Asn Val Ala Ala Ala Ile Glu Gly Arg Val Tyr 290 295 300 Ala Pro Lys Glu Val Leu Leu Lys Val Glu Glu Leu Pro Ile Val Leu 305 310 315 320 Ser Lys Leu Tyr Asn Gln Ser Lys Thr Thr Gly Lys Asp Leu Val Ala 325 330 335 Leu Ser Asp Ser Phe Val Glu Asp Lys Leu Leu Ala Tyr Thr Ser 340 345 350 8366PRTThermoactinomyces intermedius 8Met Lys Ile Phe Asp Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Met Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Cys Ile His 20 25 30 Val Thr Thr Leu Gly Pro Ala Leu Gly Gly Met Arg Met Trp Thr

Tyr 35 40 45 Ala Ser Glu Glu Glu Ala Ile Glu Asp Ala Leu Arg Leu Gly Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe 85 90 95 Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Glu 115 120 125 Glu Thr Arg Tyr Val Thr Gly Val Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Asp Asp Ser Leu Glu Gly Lys Val Val 165 170 175 Ala Val Gln Gly Val Gly His Val Ala Tyr Glu Leu Cys Lys His Leu 180 185 190 His Asn Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Asn 195 200 205 Ala Asp Arg Ala Val Gln Glu Phe Gly Ala Glu Phe Val His Pro Asp 210 215 220 Lys Ile Tyr Asp Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Ile Ile Asn Asp Glu Thr Ile Glu Arg Leu Lys Cys Lys Val Val 245 250 255 Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Glu Arg His Gly Lys Met 260 265 270 Leu Glu Glu Lys Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Leu Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Met Lys Lys Val Glu Gly Ile Tyr Asp Lys Ile Leu Lys Val 305 310 315 320 Phe Glu Ile Ala Lys Arg Asp Gly Ile Pro Ser Tyr Leu Ala Ala Asp 325 330 335 Arg Met Ala Glu Glu Arg Ile Glu Met Met Arg Lys Thr Arg Ser Thr 340 345 350 Phe Leu Gln Asp Gln Arg Asn Leu Ile Asn Phe Asn Asn Lys 355 360 365 9364PRTBacillus subtilis 9Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser 195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg 355 360 10364PRTBacillus licheniformis 10Met Glu Leu Phe Arg Tyr Met Glu Gln Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Lys Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Lys Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Val 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Ala 195 200 205 Val Glu Arg Ala Val Ala Glu Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Asp Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ser Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Lys Arg Asp Arg Ile Pro Thr Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Gln Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg 355 360 11367PRTGeobacillus stearothermophilus 11Met Glu Leu Phe Lys Tyr Met Glu Thr Tyr Asp Tyr Glu Gln Val Leu 1 5 10 15 Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Met Tyr 35 40 45 Asn Ser Glu Glu Glu Ala Leu Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe 85 90 95 Arg Ala Phe Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Ala Asp Met Asp Ile Ile Tyr Gln 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Glu Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Ala Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Ser Asp Ser Leu Glu Gly Lys Val Val 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Val 195 200 205 Val Ala Arg Ala Val Glu Glu Phe Gly Ala Lys Ala Val Asp Pro Asn 210 215 220 Asp Ile Tyr Gly Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly 225 230 235 240 Gly Ile Ile Asn Asp Gln Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ser Ala Asp Asn Gln Leu Lys Glu Pro Arg His Gly Asp Ile 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Met Lys Lys Ile Glu Gln Ile Tyr Asp Asn Ile Glu Lys Val 305 310 315 320 Phe Ala Ile Ala Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp 325 330 335 Arg Met Ala Glu Glu Arg Ile Glu Thr Met Arg Lys Ala Arg Ser Pro 340 345 350 Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg Arg Ala Arg 355 360 365 12364PRTLysinibacillus sphaericus 12Met Glu Ile Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Ala Arg Met Trp Thr Tyr 35 40 45 Ala Thr Glu Glu Asn Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Phe Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Thr Asp Met Asp Leu Ile His Glu 115 120 125 Glu Thr Asn Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Thr Asp Met Leu Glu Gly Arg Thr Ile 165 170 175 Ser Val Gln Gly Leu Gly Asn Val Ala Tyr Lys Leu Cys Glu Tyr Leu 180 185 190 His Asn Glu Gly Ala Lys Leu Val Val Thr Asp Ile Asn Gln Ala Ala 195 200 205 Ile Asp Arg Val Val Asn Asp Phe Gly Ala Thr Ala Val Ala Pro Asp 210 215 220 Glu Ile Tyr Ser Gln Glu Val Asp Ile Phe Ser Pro Cys Ala Leu Gly 225 230 235 240 Ala Ile Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ser Ala Asn Asn Gln Leu Gln Asp Ser Arg His Gly Asp Tyr 260 265 270 Leu His Glu Leu Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Leu Lys Arg Val Asp Gly Ile Tyr Asp Ser Ile Glu Lys Ile 305 310 315 320 Phe Glu Ile Ser Lys Arg Asp Ser Ile Pro Thr Tyr Val Ala Ala Asn 325 330 335 Arg Leu Ala Glu Glu Arg Ile Ala Arg Val Ala Lys Ser Arg Ser Gln 340 345 350 Phe Leu Lys Asn Glu Lys Asn Ile Leu Asn Gly Arg 355 360 13351PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(43)..(43)Any amino acid except Leu 13Met Lys Tyr Ser Leu Asn Phe Lys Glu Ile Lys Ile Asp Asp Tyr Glu 1 5 10 15 Arg Val Ile Glu Val Thr Cys Ser Lys Val Arg Leu His Ala Ile Ile 20 25 30 Ala Ile His Gln Thr Ala Val Gly Pro Ala Xaa Gly Gly Val Arg Ala 35 40 45 Ser Leu Tyr Ser Ser Phe Glu Asp Ala Cys Thr Asp Ala Leu Arg Leu 50 55 60 Ala Arg Gly Met Thr Tyr Lys Ala Ile Ile Ser Asn Thr Gly Thr Gly 65 70 75 80 Gly Gly Lys Ser Val Ile Ile Leu Pro Gln Asp Ala Pro Ser Leu Thr 85 90 95 Glu Asp Met Leu Arg Ala Phe Gly Gln Ala Val Asn Ala Leu Glu Gly 100 105 110 Thr Tyr Ile Cys Ala Glu Asp Leu Gly Val Ser Ile Asn Asp Ile Ser 115 120 125 Ile Val Ala Glu Glu Thr Pro Tyr Val Cys Gly Ile Ala Asp Val Ser 130 135 140 Gly Asp Pro Ser Ile Tyr Thr Ala His Gly Gly Phe Leu Cys Ile Lys 145 150 155 160 Glu Thr Ala Lys Tyr Leu Trp Gly Ser Ser Ser Leu Arg Gly Lys Lys 165 170 175 Ile Ala Ile Gln Gly Ile Gly Ser Val Gly Arg Arg Leu Leu Gln Ser 180 185 190 Leu Phe Phe Glu Gly Ala Glu Leu Tyr Val Ala Asp Val Leu Glu Arg 195 200 205 Ala Val Gln Asp Ala Ala Arg Leu Tyr Gly Ala Thr Ile Val Pro Thr 210 215 220 Glu Glu Ile His Ala Leu Glu Cys Asp Ile Phe Ser Pro Cys Ala Arg 225 230 235 240 Gly Asn Val Ile Arg Lys Asp Asn Leu Ala Asp Leu Asn Cys Lys Ala 245 250 255 Ile Val Gly Val Ala Asn Asn Gln Leu Glu Asp Ser Ser Ala Gly Met 260 265 270 Met Leu His Glu Arg Gly Ile Leu Tyr Gly Pro Asp Tyr Leu Val Asn 275 280 285 Ala Gly Gly Leu Leu Asn Val Ala Ala Ala Ile Glu Gly Arg Val Tyr 290 295 300 Ala Pro Lys Glu Val Leu Leu Lys Val Glu Glu Leu Pro Ile Val Leu 305 310 315 320 Ser Lys Leu Tyr Asn Gln Ser Lys Thr Thr Gly Lys Asp Leu Val Ala 325 330 335 Leu Ser Asp Ser Phe Val Glu Asp Lys Leu Leu Ala Tyr Thr Ser 340 345 350 14366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(40)..(40)Any amino acid except Leu 14Met Lys Ile Phe Asp Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Met Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Cys Ile His 20 25 30 Val Thr Thr Leu Gly Pro Ala Xaa Gly Gly Met Arg Met Trp Thr Tyr 35 40 45 Ala Ser Glu Glu Glu Ala Ile Glu Asp Ala Leu Arg Leu Gly Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe 85 90 95 Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile

His Glu 115 120 125 Glu Thr Arg Tyr Val Thr Gly Val Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Asp Asp Ser Leu Glu Gly Lys Val Val 165 170 175 Ala Val Gln Gly Val Gly His Val Ala Tyr Glu Leu Cys Lys His Leu 180 185 190 His Asn Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Asn 195 200 205 Ala Asp Arg Ala Val Gln Glu Phe Gly Ala Glu Phe Val His Pro Asp 210 215 220 Lys Ile Tyr Asp Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Ile Ile Asn Asp Glu Thr Ile Glu Arg Leu Lys Cys Lys Val Val 245 250 255 Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Glu Arg His Gly Lys Met 260 265 270 Leu Glu Glu Lys Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Leu Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Met Lys Lys Val Glu Gly Ile Tyr Asp Lys Ile Leu Lys Val 305 310 315 320 Phe Glu Ile Ala Lys Arg Asp Gly Ile Pro Ser Tyr Leu Ala Ala Asp 325 330 335 Arg Met Ala Glu Glu Arg Ile Glu Met Met Arg Lys Thr Arg Ser Thr 340 345 350 Phe Leu Gln Asp Gln Arg Asn Leu Ile Asn Phe Asn Asn Lys 355 360 365 15364PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(40)..(40)Any amino acid except Leu 15Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Xaa Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser 195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg 355 360 16364PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(40)..(40)Any amino acid except Leu 16Met Glu Leu Phe Arg Tyr Met Glu Gln Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Lys Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Xaa Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Lys Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Val 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Ala 195 200 205 Val Glu Arg Ala Val Ala Glu Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Glu Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Asp Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ser Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Lys Arg Asp Arg Ile Pro Thr Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Gln Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg 355 360 17367PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(40)..(40)Any amino acid except Leu 17Met Glu Leu Phe Lys Tyr Met Glu Thr Tyr Asp Tyr Glu Gln Val Leu 1 5 10 15 Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Xaa Gly Gly Thr Arg Met Trp Met Tyr 35 40 45 Asn Ser Glu Glu Glu Ala Leu Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe 85 90 95 Arg Ala Phe Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Ala Asp Met Asp Ile Ile Tyr Gln 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Glu Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Ala Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Ser Asp Ser Leu Glu Gly Lys Val Val 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Val 195 200 205 Val Ala Arg Ala Val Glu Glu Phe Gly Ala Lys Ala Val Asp Pro Asn 210 215 220 Asp Ile Tyr Gly Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly 225 230 235 240 Gly Ile Ile Asn Asp Gln Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ser Ala Asp Asn Gln Leu Lys Glu Pro Arg His Gly Asp Ile 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Met Lys Lys Ile Glu Gln Ile Tyr Asp Asn Ile Glu Lys Val 305 310 315 320 Phe Ala Ile Ala Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp 325 330 335 Arg Met Ala Glu Glu Arg Ile Glu Thr Met Arg Lys Ala Arg Ser Pro 340 345 350 Phe Leu Gln Asn Gly His His Ile Leu Ser Arg Arg Arg Ala Arg 355 360 365 18364PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptideMOD_RES(40)..(40)Any amino acid except Leu 18Met Glu Ile Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Xaa Gly Gly Ala Arg Met Trp Thr Tyr 35 40 45 Ala Thr Glu Glu Asn Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Phe Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Thr Asp Met Asp Leu Ile His Glu 115 120 125 Glu Thr Asn Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Glu Ala Phe Gly Thr Asp Met Leu Glu Gly Arg Thr Ile 165 170 175 Ser Val Gln Gly Leu Gly Asn Val Ala Tyr Lys Leu Cys Glu Tyr Leu 180 185 190 His Asn Glu Gly Ala Lys Leu Val Val Thr Asp Ile Asn Gln Ala Ala 195 200 205 Ile Asp Arg Val Val Asn Asp Phe Gly Ala Thr Ala Val Ala Pro Asp 210 215 220 Glu Ile Tyr Ser Gln Glu Val Asp Ile Phe Ser Pro Cys Ala Leu Gly 225 230 235 240 Ala Ile Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ser Ala Asn Asn Gln Leu Gln Asp Ser Arg His Gly Asp Tyr 260 265 270 Leu His Glu Leu Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu 290 295 300 Arg Ala Leu Lys Arg Val Asp Gly Ile Tyr Asp Ser Ile Glu Lys Ile 305 310 315 320 Phe Glu Ile Ser Lys Arg Asp Ser Ile Pro Thr Tyr Val Ala Ala Asn 325 330 335 Arg Leu Ala Glu Glu Arg Ile Ala Arg Val Ala Lys Ser Arg Ser Gln 340 345 350 Phe Leu Lys Asn Glu Lys Asn Ile Leu Asn Gly Arg 355 360 193PRTUnknownDescription of Unknown bacterial leucine dehydrogenase conserved region peptide 19Asn Val Ala 1 20366PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 20Met Thr Leu Glu Ile Phe Glu Tyr Leu Glu Lys Tyr Asp Tyr Glu Gln 1 5 10 15 Val Val Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala 20 25 30 Ile His Asp Thr Thr Leu Gly Pro Ala Ala Gly Gly Thr Arg Met Trp 35 40 45 Thr Tyr Asp Ser Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala 50 55 60 Lys Gly Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly 65 70 75 80 Ala Lys Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Ser Glu Ala 85 90 95 Met Phe Arg Ala Leu Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr 100 105 110 Ile Thr Ala Glu Asp Val Gly Thr Thr Val Asp Asp Met Asp Ile Ile 115 120 125 His Glu Glu Thr Asp Phe Val Thr Gly Ile Ser Pro Ser Phe Gly Ser 130 135 140 Ser Gly Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met 145 150 155 160 Lys Ala Ala Ala Lys Glu Ala Phe Gly Thr Asp Asn Leu Glu Gly Lys 165 170 175 Val Ile Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Lys 180 185 190 His Leu His Ala Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys 195 200 205 Glu Ala Val Gln Arg Ala Val Glu Glu Phe Gly Ala Ser Ala Val Glu 210 215 220 Pro Asn Glu Ile Tyr Gly Val Glu Cys Asp Ile Tyr Ala Pro Cys Ala 225 230 235 240 Leu Gly Ala Thr Val Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys 245 250 255 Val Ile Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Asp Arg His Gly 260 265 270 Asp Ile Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile 275 280 285 Asn Ala Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn 290 295 300 Arg Glu Arg Ala Leu Lys Arg Val Glu Ser Ile Tyr Asp Thr Ile Ala 305 310 315 320 Lys Val Ile Glu Ile Ser Lys Arg Asp Gly Ile Ala Thr Tyr Val Ala 325 330 335 Ala Asp Arg Leu Ala Glu Glu Arg Ile Ala Ser Leu Lys Asn Ser Arg 340 345 350 Ser Thr Tyr Leu Arg Asn Gly His Asp Ile Ile Ser Arg Arg 355 360 365

Claims

1-10. (canceled)

11. A nucleic acid encoding a polypeptide; wherein the polypeptide comprises: (a) the amino acid sequence of any one of SEQ ID NOS:2 and 13-18, wherein X is not leucine; (b) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, wherein X is not leucine; (c) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, wherein X is not leucine; or (d) the amino acid sequence of SEQ ID NO: 4, 5, 6, or 20; and X is isoleucine, valine, glycine, alanine, serine, or threonine.

12. An expression vector, comprising the nucleic acid of claim 11.

13. A cell, comprising the expression vector of claim 12.

14. A method for producing a polypeptide, comprising culturing the cell of claim 13 under conditions suitable for protein expression, thereby producing a polypeptide.

15. The method of claim 14, further comprising isolating the polypeptide from the cell or from media in which the cell is cultured.

16. (canceled)

17. The nucleic acid of claim 11, wherein X is isoleucine.

18. The nucleic acid of claim 11, wherein X is valine.

19. The nucleic acid of claim 11, wherein X is glycine.

20. The nucleic acid of claim 11, wherein X is alanine.

21. The nucleic acid of claim 11, wherein X is serine or threonine.

22. The nucleic acid of claim 11, wherein the amino acid sequence is SEQ ID NO:2.

23. The nucleic acid of claim 22, wherein X is alanine.

24. The expression vector of claim 12, wherein the expression vector is a prokaryotic expression vector.

25. The expression vector of claim 12, wherein the expression vector is a eukaryotic expression vector.

26. The cell of claim 13, wherein the cell is a bacterial cell.

27. The cell of claim 13, wherein the cell is a mammalian cell.

28. The cell of claim 13, wherein the cell is a yeast cell.

29. The cell of claim 13, wherein the cell is an insect cell.

30. The cell of claim 13, wherein the cell is a plant cell.

31. The method of claim 14, wherein the polypeptide comprises: (a) the amino acid sequence of any one of SEQ ID NOS:2 and 13-18, wherein X is not leucine; (b) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, wherein X is not leucine; (c) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, wherein X is not leucine; or (d) the amino acid sequence of SEQ ID NO: 4, 5, 6, or 20; and X is isoleucine, valine, glycine, alanine, serine, or threonine.