Mechanism for synthesis of isomaltooligosaccharides from maltooligosaccharides by GH15 α-glucan 4(6)-α-glucosyltransferase

Isomaltosaccharides, composed of α-(1 → 6)-d-glucosyl residues, exhibit diverse beneficial properties depending on the degree of polymerization (DP) and attract great interest across multiple industries. The anomer-retaining transglucosidase, dextran dextrinase (DDase)-which synthesizes the α-(1 → 6)-d-glucose polymer dextran from α-(1 → 4)-glucan maltooligosaccharides (MOSs)-shows structural similarity to anomer-inverting α-glucohydrolases belonging to glycoside hydrolase family 15 (GH15). Here, we show a new GH15 transglucosidase, α-glucan 4(6)-α-glucosyltransferase, from Tepidibacillus decaturensis (Td46GT) and its mechanism for converting MOS into isomaltooligosaccharide (IMO). Td46GT catalyzes DDase-like d-glucosyl-transfer reactions: α-(1 → 4)-transglucosylation to MOS and α-(1 → 6)-transglucosylation to short-chain MOS of DP 2-3 and IMO. Unlike DDase, it does not produce dextran. Kinetic analyses of the two-substrate reactions revealed that the acceptors determined formed linkages. Relative acceptor-substrate specificity constants (RASCs) indicated that maltotriose and maltose were the best acceptors for α-(1 → 4)- and α-(1 → 6)-transglucosylations, respectively. The subsite affinities calculated from the RASCs were consistent with those obtained from k_cat/K_m values for single-substrate reactions. Time-dependent changes in the MOS and IMO concentrations during the reaction were quantitatively simulated using RASCs and k_cat/K_m values. Structure prediction suggested Td46GT possesses a substrate-binding site similar to DDase, and site-directed mutagenesis identified Phe418 and Tyr612 as critical residues at subsite +2 for MOS and IMO binding. Our results suggested that Td46GT possesses distinct MOS- and IMO-binding subsites in a single active pocket, which are shared by both acceptor and donor substrates, and that the binding manner of the acceptor determines the product specificity of transglucosylation.