open access

https://hdl.handle.net/2115/98731

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

英語

alpha-glucan 4(6)-alpha-glucosyltransferase

2025-12-16

John Wiley & Sons

FEBS Journal

Isomaltosaccharides, composed of alpha-(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 alpha-(1 -> 6)-d-glucose polymer dextran from alpha-(1 -> 4)-glucan maltooligosaccharides (MOSs)-shows structural similarity to anomer-inverting alpha-glucohydrolases belonging to glycoside hydrolase family 15 (GH15). Here, we show a new GH15 transglucosidase, alpha-glucan 4(6)-alpha-glucosyltransferase, from Tepidibacillus decaturensis (Td46GT) and its mechanism for converting MOS into isomaltooligosaccharide (IMO). Td46GT catalyzes DDase-like d-glucosyl-transfer reactions: alpha-(1 -> 4)-transglucosylation to MOS and alpha-(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 alpha-(1 -> 4)- and alpha-(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.

学術雑誌論文

Accepted Manuscript

1742-464X

1742-4658