Rapid screening of the aglycone specificity of glycosidases: applications to enzymatic synthesis of oligosaccharides.
BACKGROUND
Retaining glycosidases can catalyse glycosidic bond formation through transglycosylation from a donor sugar to an acceptor bound in the aglycone site. The aglycone specificity of a glycosidase is not easily determined, thereby complicating the choice of the most appropriate glycosidase for use as a catalyst for transglycosylation. We have developed a strategy to rapidly screen the aglycone specificity of a glycosidase and thereby determine which enzymes are best suited to catalyse specific transglycosylation reactions.


RESULTS
The reactivation, or turnover, of a glycosidase trapped as a fluoroglycosyl-enzyme species is accelerated in the presence of a compound that productively binds to the aglycone site. This methodology was used to rapidly screen six glycosidases with 44 potential acceptor sugars. Validation of the screening strategy was demonstrated by the identification of products formed from a transglycosylation reaction with positively screened acceptors for four of the enzymes studied.


CONCLUSIONS
The aglycone specificity of a glycosidase can be rapidly evaluated and requires only an appropriate fluorosugar inactivator, a substrate for assay of activity and a library of compounds for screening.
Blanchard
J E
JE
Protein Engineering Network of Centres of Excellence of Canada and the Department of Chemistry, University of British Columbia, V6T 1Z1, Vancouver, BC, Canada.
Withers
S G
SG
D001426:Bacterial Proteins; D050260:Carbohydrate Metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008722:Methods; D008832:Microchemistry; D009844:Oligosaccharides / Q000096:biosynthesis; D010084:Oxidation-Reduction; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D006026:Glycoside Hydrolases