title: Mutagenesis of glycosidases.
abstract: Enzymatic hydrolysis of glycosides can occur by one of two elementary mechanisms identified by the stereochemical outcome of the reaction, inversion or retention. The key active-site residues involved are a pair of carboxylic acids in each case, and strategies for their identification and for probing the details of their roles in catalysis have been developed through detailed kinetic analysis of mutants. Similarly the roles of other active-site residues have also been probed this way, and mutants have been developed that trap intermediates in catalysis, allowing the determination of the three-dimensional structures of several such key species. By manipulating the locations or even the presence of these carboxyl side chains in the active site, the mechanisms of several glycosidases have been completely changed, and this has allowed the development of "glycosynthases," mutant glycosidases that are capable of synthesizing oligosaccharides but unable to degrade them. Surprisingly little progress has been made on altering specificities through mutagenesis, although recent results suggest that gene shuffling coupled with effective screens will provide the most effective approach.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, Canada.
authors.forename: H D
authors.initials: HD
authors.lastname: Ly
authors.forename: S G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D013379:Substrate Specificity
chemical_list: D006026:Glycoside Hydrolases