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 . A mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate amino acid residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif E or SEQ ID NO:46) of a corresponding wild-type endoglycoceramidase, wherein the mutant endoglycoceramidase catalyzes the transfer of a saccharide moiety from a donor substrate to an acceptor substrate. 
     
     
         2 . The mutant endoglycoceramidase of  claim 1 , wherein the wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (motif D or SEQ ID NO:45). 
     
     
         3 . The mutant endoglycoceramidase of  claim 1 , wherein the synthetic activity is increased compared to that of the corresponding wild-type endoglycoceramidase. 
     
     
         4 . The mutant endoglycoceramidase of  claim 1 , wherein the hydrolytic activity is decreased compared to that of the corresponding wild-type endoglycoceramidase. 
     
     
         5 . The mutant endoglycoceramidase of  claim 2 , wherein the nucleophilic Glu residue is substituted with an amino acid other than Glu. 
     
     
         6 . The mutant endoglycoceramidase of  claim 5 , wherein the nucleophilic Glu residue is substituted with an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val. 
     
     
         7 . The mutant endoglycoceramidase of  claim 1 , wherein the enzyme has had its native signal peptide sequence removed. 
     
     
         8 . The mutant endoglycoceramidase of  claim 4 , wherein the corresponding wild-type endoglycoceramidase is selected from the group consisting of a  Rhodococcus  endoglycoceramidase, a  Propionibacterium  endoglycoceramidase, a  Dictyostelium  endoglycoceramidase, a  Cyanea  endoglycoceramidase, a  Hydra  endoglycoceramidase, a  Streptomyces  endoglycoceramidase, a  Schistosoma  endoglycoceramidase, a  Leptospira  endoglycoceramidase, and a  Neurospora  endoglycoceramidase. 
     
     
         9 . The mutant endoglycoceramidase of  claim 2 , wherein the corresponding wild-type endoglycoceramidase comprises an amino acid sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16-20. 
     
     
         10 . The mutant endoglycoceramidase of  claim 1 , wherein the mutant endoglycoceramidase is part of a fusion protein. 
     
     
         11 . The mutant endoglycoceramidase of  claim 1 , further comprising an amino acid tag. 
     
     
         12 . The mutant endoglycoceramidase of  claim 1 , further comprising an N-terminus (Met/Val/Leu)-Leu-Asp-(Met-Phe-Ala)-His-Gln-Asp-(Met/Val/Leu)-X-(Ser/Asn) motif (motif A or SEQ ID NO:42) and a C-terminus Ala-Ile-Arg-(Gln/Ser/Thr)-Val-Asp motif (motif C or SEQ ID NO:44). 
     
     
         13 . A nucleic acid that comprises a nucleotide sequence that encodes a mutant endoglycoceramidase of  claim 1 . 
     
     
         14 . An expression vector that comprises the nucleic acid sequence of  claim 13 . 
     
     
         15 . A host cell that comprises the expression vector of  claim 14 . 
     
     
         16 . A method of producing a mutant endoglycoceramidase, the method comprising growing the host cell of  claim 15 , under conditions suitable for expression of the mutant endoglycoceramidase. 
     
     
         17 . A mutant endoglycoceramidase comprising an amino acid sequence of any one of SEQ ID NOs:47-58. 
     
     
         18 . A method for making a mutant endoglycoceramidase having enhanced synthetic activity in comparison to a corresponding wild-type endoglycoceramidase, the method comprising modifying the nucleophilic carboxylate amino acid residue in a corresponding wild-type endoglycoceramidase, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (SEQ ID NO:46) of a corresponding wild-type endoglycoceramidase. 
     
     
         19 . The method of  claim 18 , wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (SEQ ID NO:45) of a corresponding wild-type endoglycoceramidase. 
     
     
         20 . The method of  claim 19 , wherein the nucleophilic Glu residue is modified by replacing the codon encoding the nucleophilic Glu residue with a codon encoding an amino acid other than Glu. 
     
     
         21 . The method of  claim 20 , wherein the nucleic acid codon encoding the nucleophilic Glu residue is replaced with a nucleic acid codon encoding an amino acid residue selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val. 
     
     
         22 . A method of synthesizing a glycolipid, the method comprising, contacting a donor substrate comprising a saccharide moiety and an acceptor substrate with a mutant endoglycoceramidase having a modified nucleophilic carboxylate amino acid residue, wherein the nucleophilic carboxylate residue resides within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-(Glu/Asp)-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (SEQ ID NO:46) of a corresponding wild-type endoglycoceramidase, under conditions wherein the endoglycoceramidase catalyzes the transfer of a saccharide moiety from a donor substrate to an acceptor substrate, thereby producing the glycolipid. 
     
     
         23 . The method of  claim 22 , wherein the nucleophilic carboxylate amino acid residue is a Glu residing within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence (SEQ ID NO:45) of a corresponding wild-type endoglycoceramidase. 
     
     
         24 . The method of  claim 22 , wherein the donor substrate is an α-modified glycosyl donor of anomeric configuration opposite the natural glycosidic linkage. 
     
     
         25 . The method of  claim 24 , wherein the donor substrate is a glycosyl fluoride. 
     
     
         26 . The method of  claim 22 , wherein the acceptor substrate is an aglycone of Formulas Ia, Ib, II or III. 
     
     
         27 . The method of  claim 26 , wherein the acceptor substrate is a sphingosine or a sphingosine analog. 
     
     
         28 . The method of  claim 27 , wherein the sphingosine is selected from the group consisting of from D-erythro-sphingosine, D-erythro-sphinganine, L-threo-sphingosine, L-threo-dihydrosphingosine, D-erythro-phytosphingosine, N-ocatanoyl-D-erythro-sphingosine. 
     
     
         29 . The method of  claim 26 , wherein the acceptor substrate is a ceramide. 
     
     
         30 . The method of  claim 22 , wherein the glycolipid is selected from the group consisting of a glycosphingolipid, a ganglioside and a cerebroside. 
     
     
         31 . The method of  claim 30 , wherein the glycolipid is a ganglioside selected from the group consisting of GD 1a , GD 1α , GD 1b , GD 2 , GD 3 , Gg3, Gg4, GH 1 , GH 2 , GH 3 , GM 1 , GM 1b , GM 2 , GM 3 , Fuc-GM 1 , GP 1 , GP 2 , GP 3 , GQ 1b , GQ 1B , GQ 1β , GQ 1c , GQ 2 , GQ 3 , GT 1a , GT 1b , GT 1c , GT 1β , GT 1c , GT 2 , GT 3 , and polysialylated lactose. 
     
     
         32 . A reaction mixture comprising a mutant endoglycoceramidase of  claim 1 , a donor substrate comprising a saccharide moiety and an acceptor substrate. 
     
     
         33 . The reaction mixture of  claim 32 , wherein the acceptor substrate is a ceramide, a sphingosine or a sphingosine analog. 
     
     
         34 . A mutant endoglycoceramidase characterized in that
 i) in its native form the endoglycoceramidase comprises an amino acid sequence that is any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16-20; and   ii) the nucleophilic Glu residue within a (Ile/Met/Leu/Phe/Val)-(Leu/Met/Ile/Val)-(Gly/Ser/Thr)-Glu-(Phe/Thr/Met/Leu)-(Gly/Leu/Phe) sequence of a corresponding wild-type endoglycoceramidase is modified to an amino acid other than Glu.   
     
     
         35 . The mutant endoglycoceramidase of  claim 34 , wherein the nucleophilic Glu residue is modified to an amino acid selected from the group consisting of Gly, Ala, Ser, Asp, Asn, Gln, Cys, Thr, Ile, Leu and Val.