Mutational analysis of glycosylase function: A mini-review

Enzymes catalyzing transfer of glycosyl groups to water (glycosidases), or carbohydrate OH-groups (glycosyl transferases) utilize general acid catalysis to cleave the glycosidic linkage. Action on α- or β-glycosidic bonds with either retention or inversion of the anomeric configuration represents variations in mechanism. The recent determination of three-dimensional structures of several glycosylase-substrate analogue complexes has, together with kinetic analysis of mutant enzymes, improved the knowledge on the mechanisms of action. The present review compiles and discusses information on glycosylase function gained from mutants of 33 enzymes (representing 15 EC entries), most of which are produced by directed mutagenesis of the corresponding cDNAs. These results help identify essential groups and their role in catalysis. In general, replacement of the catalytic general acid (Asp, Glu, or Tyr) results in inactivation, while low activity often remains after mutation of other residues involved in catalysis. The participation in substrate recognition and binding, transition state stabilization and product specificity has been addressed for other active site residues. Enhancement of activity and change of the mechanistic pathway have been reported for a few enzymes. Together these results provide a basis for engineering glycosylase properties using genetic techniques.