Discovery of a Novel Microbial Glycoside Hydrolase Directing Formation of New α-Amylase Inhibitors

Glycoside hydrolases (GHs) comprise a large and diverse family of enzymes that catalyze the hydrolysis of glycosidic bonds in a wide range of glycan substrates, including polysaccharides, oligosaccharides, and glycoconjugates. In addition to their fundamental roles in biological systems, GHs are widely exploited in industrial and biomedical applications. Here, we report the identification and functional characterization of a new GH, Acm11, from the biosynthetic gene cluster of pseudo-oligosaccharide acarviostatin I03 in a streptomyce strain. Bioinformatic analysis predicts Acm11 to be an α-amylase containing a C-terminal starch-binding domain. Disruption of the acm11 resulted in decrease of acarviostatin I03 production. Consistently, in vitro enzymatic assays demonstrated that Acm11 efficiently hydrolyzes starch, indicating its involvement in acarviostatin I03 biosynthesis. Notably, Acm11 also exhibited hydrolytic activity toward oligosaccharide substrates, progressively removing glucose units from acarviostatin I03 and acarbose. In addition to hydrolysis, Acm11 catalyzed C7-cyclitol-(1→4)-glycosylation of acarviostatin I03 and acarbose in the presence of maltooligosaccharides, revealing an unexpected transglycosylation activity. Mutational analysis indicated that distinct amino acid residues are responsible for hydrolytic and transglycosylation activities and evolution of Acm11 is expected to yield variants with altered catalytic preferences as the promising biocatalysts to generate structurally diverse and functionally improved α-amylase inhibitors.