Mutation of a Conserved Tryptophan in the Chitin-Binding Cleft of Serratia marcescens Chitinase A Enhances Transglycosylation

Abstract

Family 18 chitinases have the signature peptide DGXDXDXE forming the fourth β-strand in the (β⁄α)₈-barrel of their catalytic domain. The carboxyl-end glutamic acid, E315 in Serratia marcescens chitinase A, serves as the acid/base during chitin hydrolysis, and the side-chain of the preceding aspartic acid, D313, helps to position correctly the N-acetyl moiety of the glycosyl sugar undergoing hydrolysis. Chitin substrates are bound within a long cleft across the top of the barrel, whose floor consists of aromatic residues that hydrophobically stack with every other GlcNAc. Alanine substitution of the conserved Trp167 at the −3 subsite in Serratia marcescens chitinase A enhanced transglycosylation. Higher oligosaccharides were formed from both chitin tetra- and pentasaccharide, and the only hydrolytic product from chitin trisaccharide was the disaccharide. Greater retention of the glycosyl fragment at the active site of the −3 mutant of Serratia marcescens chitinase A might favor transglycosylation due to a stabilized conformation of its D313.