Mutant endoglycoceramidases with enhanced synthetic activity

Abstract

The present invention relates to a novel endoglycoceramidase whose hydrolytic activity has been substantially reduced or eliminated, such that the enzyme is useful for synthesis of glycolipids from a monosaccharide or oligosaccharide and a ceramide. More specifically, the endoglycoceramidase is a mutant version of a naturally occurring endoglycoceramidase, preferably comprising a mutation within the active site or the nucleophilic site of the enzyme and more preferably comprising a substitution mutation of the Glu residue within the active site or the nucleophilic site. Also disclosed are a method for generating the mutant endoglycoceramidase and a method for enzymatically synthesizing glycolipids using this mutant enzyme.

1 . (canceled) 
     
     
         2 . A mutant endoglycoceramidase, wherein the mutant endoglycoceramidase comprises a wild-type endoglycoceramidase peptide sequence including a nucleophilic region as set forth in SEQ ID NO: 54 modified by replacing a nucleophilic carboxylate amino acid residue of the nucleophilic region with a serine (Ser), glycine (Gly), or alanine (Ala) amino acid residue. 
     
     
         3 . The mutant endoglycoceramidase of  claim 2 , wherein the mutant is capable of catalyzing the transfer of a saccharide moiety from a donor substrate to an acceptor substrate selected from a sphingosine, a ceramide, or an analog thereof, thereby producing a glycolipid. 
     
     
         4 . The mutant endoglycoceramidase of  claim 2 , wherein the corresponding wild-type endoglycoceramidase comprises an amino acid sequence set forth in SEQ ID NOs: 3, 6, 8, 11, 14, 17, 20, 23, 25, 26, 27, or 28. 
     
     
         5 . The mutant endoglycoceramidase of  claim 2 , comprising any one of the amino acid sequences set forth in SEQ ID NOS: 55-66. 
     
     
         6 . A nucleic acid encoding the mutant endoglycoceramidase of  claim 2 . 
     
     
         7 . A vector comprising the nucleic acid of  claim 6 . 
     
     
         8 . A host cell comprising the nucleic acid of  claim 6 . 
     
     
         9 . A host cell comprising the vector of  claim 7 . 
     
     
         10 . A method of producing a mutant endoglycoceramidase, comprising growing the host cell of  claim 8  under conditions suitable for expression of the mutant endoglycoceramidase. 
     
     
         11 . A host cell expressing the mutant endoglycoceramidase of  claim 2 . 
     
     
         12 . The mutant endoglycoceramidase of  claim 3 , wherein the mutant catalyzes the transfer of the saccharide moiety from the donor substrate to the acceptor substrate at a rate that exceeds hydrolysis of the glycolipid. 
     
     
         13 . The mutant endoglycoceramidase of  claim 3 , wherein the mutant exhibits increased catalytic activity in the transfer of the saccharide moiety from the donor substrate to the acceptor substrate as compared to the wild-type endoglycoceramidase. 
     
     
         14 . The mutant endoglycoceramidase of  claim 3 , wherein the mutant exhibits decreased catalytic activity in hydrolyzing the glycolipid as compared to the wild-type endoglycoceramidase. 
     
     
         15 . A method of producing a glycolipid, the method comprising: contacting a donor substrate comprising an activated saccharide moiety and an aglycone acceptor substrate with the mutant endoglycoceramidase of  claim 2  in a reaction mixture. 
     
     
         16 . The method of  claim 15 , wherein the mutant catalyzes the transfer of the saccharide moiety from the donor substrate to the acceptor substrate at a rate that exceeds hydrolysis of the glycolipid. 
     
     
         17 . The method of  claim 15 , wherein the mutant endoglycoceramidase exhibits increased catalytic activity in the transfer of the saccharide moiety from the donor substrate to the acceptor substrate as compared to the wild-type endoglycoceramidase. 
     
     
         18 . The method of  claim 15 , wherein said mutant exhibits decreased catalytic activity in hydrolyzing the glycolipid as compared to the wild-type endoglycoceramidase. 
     
     
         19 . The method of  claim 15 , wherein the acceptor substrate is sphingosine. 
     
     
         20 . The method of  claim 15 , wherein the acceptor substrate is a sphingosine analog having a structure defined by the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         Z is O, S, C(R 2 ) 2  or NR 2 ; 
         X is H, —OR 3 , —NR 3 R 4 , CR 3 , or —CHR 3 R 4 ; 
         R 1 , R 2 , R 3  and R 4  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, —C(=M)R 5 , —C(=M)-Z 1 —R 5 , —SO 2 R 5 , or —SO 3 ; wherein 
         M and Z 1  are independently selected from O, NR 6  or S; and 
         R 5 , R 6 , R 7  and R 8  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl; 
         Y is H, —OR 7 , —SR 7 , —NR 7 R 8 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; and 
         R a , R b , R c  and R d  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl. 
       
     
     
         21 . The method of  claim 15 , wherein the acceptor substrate is a sphingosine analog selected from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, and N-ocatanoyl-D-erythro-sphingosine. 
     
     
         22 . The method of  claim 15 , wherein the acceptor substrate is a molecule having a structure selected from: 
       
         
           
           
               
               
           
         
       
     
     
         23 . The method of  claim 15 , wherein the donor substrate is a glycosyl fluoride. 
     
     
         24 . The method of  claim 15 , wherein the glycolipid is a ganglioside selected from disialoganglioside (GD 1a , GD 1α , GD 1b , GD 2 , GD 3 ), galactosylganglioside (Gg 3 , Gg 4 ), monosialyltetrahexosylceramide (GH 1 , GH 2 , GH 3 ), monosialoganglioside (GM 1 , GM 1b , GM 2 , GM 3 , Fuc-GM 1 ), pentasialoganglioside (GP 1 , GP 2 , GP 3 ), tetrasialoganglioside (GQ 1b , GQ 1B , GQ 1β , GQ 1c , GQ 2 , GQ 3 ), trisialoganglioside (GT 1a , GT 1b , GT 1c , GT 1β , GT 1c , GT 2 , and GT 3 ). 
     
     
         25 . The method of  claim 15 , wherein the glycolipid is monosialoganglioside 1 (GM 1 ). 
     
     
         26 . The method of  claim 25 , wherein the glycolipid is lysomonosialoganglioside 1 (lyso-GM1).