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

Understanding what amino acids in alpha-1,6-glucanases target alpha-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 alpha-1,6 glycosidic bonds. Compu-tational analysis identified two amino acids, E343 and W521, trigger alpha-1,6 glycosidic bond specificity of en-zymes. 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 obvi-ously 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).

Automated summary

This study investigated the amino acids involved in the recognition and catalysis of alpha-1,6 glycosidic bonds, and identified E343 and W521 as crucial residues for alpha-1,6-glucanases. By studying engineered mutants, it was found that these amino acids significantly affected the product structure. The catalytic mechanism revealed that bulky aromatic side chains controlled the ratio of branch glucans, and E148 acted as a proton donor to regulate the generation of branched structures.