Glycosyltransferase reversibility for sugar nucleotide synthesis
The present invention generally relates to materials and methods for exploiting glycosyltransferase reversibility for nucleotide diphosphate (NDP) sugar synthesis. The present invention provides engineered glycosyltransferase enzymes characterized by improved reaction reversibility and expanded sugar donor specificity as compared to corresponding non-mutated glycosyltransferase enzymes. Such reagents provide advantageous routes to NDP sugars for subsequent use in a variety of biomedical applications, including enzymatic and chemoenzymatic glycorandomization.
1. An isolated mutant glycosyltransferase comprising:
(a) the amino acid sequence of OleD glycosyltransferase set forth in SEQ ID NO: 1, wherein proline at position 67 has been replaced with threonine, serine at position 132 has been replaced with phenylalanine, alanine at position 242 has been replaced with leucine, and glutamine at position 268 has been replaced with valine; or
(b) an amino acid sequence which has at least 95% sequence identity to SEQ ID NO:1, wherein the proline at position 67 has been replaced with threonine, the serine at position 132 has been replaced with phenylalanine, the alanine at position 242 has been replaced with leucine, and the glutamine at position 268 has been replaced with valine,
wherein said isolated mutant exhibits an improved conversion of nucleotide diphosphate (NDP) to NDP sugar as compared to a corresponding non-mutated glycosyltransferase.
2. The isolated mutant glycosyltransferase according to claim 1 , wherein said isolated mutant glycosyltransferase is encoded by a nucleotide that hybridizes under stringent conditions 4× sodium chlorine/sodium citrate (SSC), at about 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. to the nucleotide sequence set forth in SEQ ID NO:2.