Enzymatic and Structural Characterization of a Branching Enzyme FdBE2 from the Gut Bacterium Faecalibacterium duncaniae

Abstract

Branching enzyme catalyzes transglucosylation on α-1,4-glucan chains in glycogen and starch to form α-1,6-glucosidic linkages. Bacterial branching enzymes are classified into subfamily 9 of glycoside hydrolase family 13 (GH13_9). Faecalibacterium duncaniae, a butyrate-producing gut bacterium, has two genes encoding GH13_9 enzymes (protein IDs: GXM22_01075 and GXM22_04345), which were designated here as FdBE1 and FdBE2, respectively. Here, recombinant FdBE2 was expressed in Escherichia coli and purified. The enzyme showed branching enzyme activity toward amylose and an optimal pH of 6.0-7.0. Analysis of reaction products revealed that FdBE2 predominantly generated branched glucan chains ranging in degree of polymerization (DP) from 6 to 17, with DP7 as the most abundant product. Crystal structures of unliganded FdBE2 and its maltohexaose complex were determined. FdBE2 consists of a CBM48 domain followed by domains A, B, and C common to GH13 enzymes. The crystal structure of FdBE2 in complex with maltohexaose indicated that two maltohexaose molecules bind to its molecular surface. One of the maltooligosaccharide-binding sites was unique to FdBE2, suggesting that although numerous maltooligosaccharides can bind to the molecular surface of GH13_9 enzymes, their binding patterns are diverse. The results here suggest that FdBE2 is a distinctive enzyme with characteristics different from previously reported GH13_9 branching enzymes.