Chemically modified mutant serine hydrolases show improved catalytic activity and chiral selectivity

Abstract

This invention provides novel chemically modified mutant serine hydrolases that catalyze a transamidation and/or a transpeptidation and/or a transesterification reaction. The modified serine hydrolases have one or more amino acid residues in a subsite replaced with a cysteine, wherein the cysteine is modified by replacing the thiol hydrogen in the cysteine with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, and a glycoside. In particularly preferred embodiments, the substitutents include an oxazolidinone, a C 1 to C 15 alkyl amino group with a positive charge, or a glycoside.

1 . A modified serine hydrolase that catalyzes a transamidation or a transpeptidation or a transesterification reaction, said protease having one or more amino acid residues in a subsite replaced with a cysteine, wherein the cysteine is modified by replacing the thiol hydrogen in the cysteine with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, a chiral substituent, a heterocyclic substituent, and a glycoside.  
   
   
       2 . The modified serine hydrolase of  claim 1 , wherein the serine hydrolase catalyzes a transamidation.  
   
   
       3 . The modified serine hydrolase of  claim 1 , wherein the serine hydrolase catalyzes a transpeptidation.  
   
   
       4 . The modified serine hydrolase of  claim 1 , wherein the serine hydrolase catalyzes a transesterification.  
   
   
       5 . The modified serine hydrolase of  claim 1 , wherein said serine hydrolase is selected from the group consisting of an alpha/beta serine hydrolase, a subtilisin type serine protease, and a chymotrypsin serine protease.  
   
   
       6 . The modified serine hydrolase of  claim 1 , wherein said serine hydrolase is a subtilisin.  
   
   
       7 . The modified serine hydrolase of  claim 6 , wherein said serine hydrolase catalyzes a transamidation and is stereoselective.  
   
   
       8 . The modified serine hydrolase of  claim 6 , wherein the amino acid replaced with a cysteine is an amino acid in the S 1 , S 1 ′, or S 2  subsite.  
   
   
       9 . The modified serine hydrolase of  claim 8 , wherein the amino acid replaced with a cysteine is selected from the group consisting of asparagine, leucine, methionine, and serine.  
   
   
       10 . The modified serine hydrolase of  claim 8 , wherein said amino acid is selected from the group consisting of amino acid 156 in the S 1  subsite, amino acid 166 in the S 1  subsite, amino acid 217 in the S 1 ′ subsite, amino acid 222 in S 1 ′ subsite and amino acid 62 in the S2 subsite.  
   
   
       11 . The modified serine hydrolase of  claim 1 , wherein said substitutent is selected from the group consisting of an oxazolidinone, a C 1  to C 15  alkyl amino group with a positive charge, and a glycoside.  
   
   
       12 . The modified serine hydrolase of  claim 11 , wherein said glycoside is selected from the group consisting of a monosaccaharide, a disaccharides, and an oligosaccharide comprising pentoses and hexoses.  
   
   
       13 . The modified serine hydrolase of  claim 1 , wherein said substitutent is selected from the group consisting of the substituents listed in  FIG. 2 .  
   
   
       14 . The modified serine hydrolase of  claim 1 , wherein said substitutent is selected from the group consisting of (R)-2-methoxy-2-phenyl-ethyl-thiol, (S)-2-methoxy-2-phenyl-ethyl-thiol, (R)-2-hydroxy-2-phenyl-ethyl-thiol, (S)-2-hydroxy-2-phenyl-ethyl-thiol, N-(3′-thio-propyl)-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-phenyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(R)-4phenyul-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-phenyl-2-oxazolidinone, N-(2′-thioethyl)-(R)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-benzyl-2-oxazolidinone, N-(3′-thio)-(3aR-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one, and N-(3′-thio)-(3aS-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one.  
   
   
       15 . A chemically modified mutant subtilisin, said subtilisin having one or more amino acid residues selected from the S 1 , S 1 ′, or S 2  subsites replaced with a cysteine, wherein the cysteine is modified by replacing the thiol hydrogen in the cysteine with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, an alkyl group bearing a negatively charged moiety, and a glycoside.  
   
   
       16 . The subtilisin of  claim 15 , wherein the amino acid residue replaced with a cysteine is selected from the group consisting of amino acid 62, amino acid 156, amino acid 166, amino acid 217, and amino acid 222.  
   
   
       17 . The subtilisin of  claim 16 , wherein said substitutent is selected from the group consisting of an oxazolidinone, a C 1  to C 15  alkyl amino group with a positive charge, a C 1  to C 15 —SO 3   − , C 1  to C 15 —CO 2   − , and a glycoside.  
   
   
       18 . The subtilisin of  claim 17 , wherein said glycoside is selected from the group consisting of a monosaccaharide, a disaccharides, an oligosaccharide comprising pentoses and hexoses.  
   
   
       19 . The subtilisin of  claim 16 , wherein said substitutent is selected from the group consisting of the substituents listed in  FIG. 2 .  
   
   
       20 . The subtilisin of  claim 16 , wherein said substitutent is selected from the group consisting of (R)-2-methoxy-2-phenyl-ethyl-thiol, (S)-2-methoxy-2-phenyl-ethyl-thiol, (R)-2-hydroxy-2-phenyl-ethyl-thiol, (S)-2-hydroxy-2-phenyl-ethyl-thiol, N-(3′-thio-propyl)-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-phenyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(R)-4phenyul-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-phenyl-2-oxazolidinone, N-(2′-thioethyl)-(R)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-benzyl-2-oxazolidinone, N-(3′-thio)-(3aR-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one, and N-(3′-thio)-(3aS-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one.  
   
   
       21 . A method of forming a peptide bond, said method comprising 
 contacting a compound comprising an ester substrate with a serine hydrolase of  claim 1  or  15  and a primary amine under conditions whereby said hydrolase catalyzes the formation of a peptide bond.    
   
   
       22 . The method of  claim 21 , wherein said compound comprising an ester substrate is an acyl donor and said primary amine is an acyl acceptor.  
   
   
       23 . The method of  claim 22 , wherein said acyl acceptor is an amino acid amide.  
   
   
       24 . The method of  claim 23 , wherein said amino acid amide is present in a peptide.  
   
   
       25 . The method of  claim 22 , wherein said acyl acceptor is an L-amino acid amide.  
   
   
       26 . The method of  claim 22 , wherein said acyl acceptor is a D-amino acid amide.  
   
   
       27 . The method of  claim 22 , wherein said ester substrate is an amino acid ester.  
   
   
       28 . The method of  claim 27 , wherein said amino acid ester is present in a peptide.  
   
   
       29 . The method of  claim 22 , wherein said ester substrate is an L-amino acid ester.  
   
   
       30 . The method of  claim 22 , wherein said ester substrate is a D-amino acid ester.  
   
   
       31 . A method of resolving racemic primary and secondary alcohols using a transesterification reaction, said method comprising contacting said racemic primary or secondary alcohols with a serine hydrolase of claims  1  or  15  and an acyl donor whereby said serine hydrolase catalyzes a transesterification reaction resolving said recemic primary or secondary alcohol.  
   
   
       32 . The method of  claim 31 , wherein said primary or secondary alcohol is selected from the group consisting of an aliphatic alcohol, an aromatic alcohol, and a heterocyclic alcohol.  
   
   
       33 . The method of  claim 31 , wherein said primary or secondary alcohol is selected from the group consisting of 2-phenyl-1-propanol, 2-methyl-1-pentanol, and 2 octanol.  
   
   
       34 . The method of  claim 31 , wherein said acyl donors are selected from the group consisting of carboxylic acid esters and activated esters.  
   
   
       35 . The method of  claim 34 . wherein said carboxylic acid esters are selected from the group consisting of alkyl carboxylic esters, and aralkyl esters.  
   
   
       36 . The method of  claim 34 . wherein said activated ester is selected from the group consisting of a monohaloalkyl, a dihaloalkyl, and a trihaloalkyl.  
   
   
       37 . The method of  claim 31 , wherein said modified mutant enzyme is selected from the group consisting of L217C—(CH 2 ) 2 —SO 3   − , N62C— (CH 2 ) 2 —SO 3   − , and N62C—S—CH 3 .  
   
   
       38 . A method of attaching a chiral moiety to a substrate via a transamidation, a transesterification, or a transpeptidation reaction, said method comprising contacting said substrate having a reactive site suitable for a transesterification or a tansamidation, and said moiety with a catalytic serine hydrolase of claims  1  or  15  under conditions whereby said chiral moiety is covalently coupled to said substrate.  
   
   
       39 . The method of  claim 38 , wherein said moiety is a chiral is selected from the group consisting of a D amino acid, an L-amino acid, an acyclic aliphatic, a cyclic aliphatic, an aralkyl. R-carboxylic acid, and aralkyl S-carboxylic acid, an aromatic R-carboxylic acid, and an aromatic S-carboxylic acid.  
   
   
       40 . The method of  claim 39 , wherein said reaction is preferential for a moiety of one chirality.  
   
   
       41 . The method of  claim 39 , wherein said transesterification results in an enantiomerically biased product.  
   
   
       42 . The method of  claim 38 , wherein said substrate is an amino acid or a polypeptide.  
   
   
       43 . A method of incorporating an amino acid into a polypeptide, said method comprising contacting an amino acid ester with a catalytic serine protease of  claim 1  or  15  and an amino acid primary amine under conditions whereby said serine hydrolase catalyzes the formation of a peptide bond between the amino acid of said amino acid ester and the amino acid of the amino acid amine.  
   
   
       44 . The method of  claim 43 , wherein said amino acid ester is an acyl donor and said amino acid amine is an acyl acceptor.  
   
   
       45 . The method of  claim 43 , wherein said amino acid amide is present in a peptide.  
   
   
       46 . The method of  claim 45 , wherein said amino acid amide is an L-amino acid amide.  
   
   
       47 . The method of  claim 45 , wherein said amino acid amide is a D-amino acid amide.  
   
   
       48 . The method of  claim 43 , wherein said amino acid ester is an L-amino acid ester.  
   
   
       49 . The method of  claim 43 , wherein said amino acid ester is a D-amino acid ester.  
   
   
       50 . The method of  claim 43 , wherein said amino acid ester is present in a peptide.  
   
   
       51 . A method of producing a chemically modified mutated serine hydrolase, said method comprising 
 providing a serine hydrolase wherein one or more amino acids have been replaced with cysteine residues; and    replacing the thiol hydrogens in the cysteine residues with a substituent group providing a thiol side chain comprising a moiety selected from the group consisting of consisting of a polar aromatic substituent, an alkyl amino group with a positive charge, and a glycoside.    
   
   
       52 . The method of  claim 51 , wherein said hydrolase is selected from the group consisting of an alpha/beta serine protease, a subtilisin type serine protease, and a chymotrypsin serine protease.  
   
   
       53 . The method of  claim 51 , wherein said hydrolase is a subtilisin.  
   
   
       54 . The method of  claim 53 , wherein the amino acid replaced with a cysteine is an amino acid in the S 1 , S 1 ′, or S 2  subsite.  
   
   
       55 . The method of  claim 53 , wherein the amino acid replaced with a cysteine is selected from the group consisting of asparagine, leucine, methionine, and serine.  
   
   
       56 . The method of  claim 53 , wherein said amino acid is selected from the group consisting of amino acid 156 in the S 1  subsite, amino acid 166 in the S 1  subsite. amino acid 217 in the S 1 ′ subsite, amino acid 222 in S 1 ′ subsite and amino acid 62 in the S2 subsite.  
   
   
       57 . The method of  claim 53 , wherein said substitutent is selected from the group consisting of an oxazolidinone, a C 1  to C 15  alkyl amino group with a positive charge, and a glycoside.  
   
   
       58 . The method of  claim 57 , wherein said glycoside is selected from the group consisting of a monosaccaharide, a disaccharides, and an oligosaccharide comprising pentoses and hexoses.  
   
   
       59 . The method of  claim 53 , wherein said substitutent is selected from the group consisting of the substituents listed in  FIG. 2 .  
   
   
       60 . The method of  claim 53 , wherein said substitutent is selected from the group consisting of (R)-2-methoxy-2-phenyl-ethyl-thiol, (S)-2-methoxy-2-phenyl-ethyl-thiol, (R)-2-hydroxy-2-phenyl-ethyl-thiol, (S)-2-hydroxy-2-phenyl-ethyl-thiol, N-(3′-thio-propyl)-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-phenyl-2-oxazolidinone, N-(3′-thio-propyl)-(R)-4-benzyl-2-oxazolidinone, N-(3′-thio-propyl)-(S)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(R)-4phenyul-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-phenyl-2-oxazolidinone, N-(2′-thioethyl)-(R)-4-benzyl-2-oxazolidinone, N-(2′-thio-ethyl)-(S)-4-benzyl-2-oxazolidinone, N-(3′-thio)-(3aR-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one, and N-(3′-thio)-(3aS-cis)-3,3a,8,8a-tetrahydro-2H-indeno[1,2-d]-oxazol-2-one.  
   
   
       61 . The method of  claim 53 , wherein said method further comprises screening the modified serine hydrolase for an activity selected from the group consisting of a transesterification activity, a transamidation activity, and a transpeptidation activity.  
   
   
       62 . The method of  claim 61 , wherein said activity is stereoselective.