MUTAGENESIS OF GLYCOSIDASES

• Published in: Annual review of biochemistry Vol. 68; no. 1; pp. 487 - 522
• Main Authors: Ly, Hoa D, Withers, Stephen G
• Format: Journal Article
• Language: English
• Published: Palo Alto, CA 94303-0139 Annual Reviews 01-01-1999; 4139 El Camino Way, P.O. Box 10139 Annual Reviews, Inc; USA
• Subjects: Carboxylic acids; Catalysis; glycosidase; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; glycosides; glycosyl transfer; hydrolase; mechanism; Mutagenesis; mutation; oligosaccharides; Substrate Specificity
• ISSN: 0066-4154; 1545-4509
• DOI: 10.1146/annurev.biochem.68.1.487

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.