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 .- 50 . (canceled) 
     
     
         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 monosaccharide, 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)-4-phenyul-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)-3,3-a,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.