Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product

Abstract

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).