Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering

Alternansucrase (ALT, EC 2.4.1.140) is a glucansucrase that can generate α-(1,3/1,6)-linked glucan from sucrose. Previously, the crystal structure of the first alternansucrase from Leuconostoc citreum NRRL B-1355 was successfully elucidated; it showed that alternansucrase might have two acceptor subsites (W675 and W543) responsible for the formation of alternating linked glucan. This work aimed to investigate the primary acceptor subsite (W675) by saturated mutagenesis using Leuconostoc citreum ABK-1 alternansucrase (LcALT). The substitution of other residues led to loss of overall activity, and formation of an alternan polymer with a nanoglucan was maintained when W675 was replaced with other aromatic residues. Conversely, substitution by nonaromatic residues led to the synthesis of oligosaccharides. Mutations at W675 could potentially cause LcALT to lose control of the acceptor molecule binding via maltose–acceptor reaction—as demonstrated by results from molecular dynamics simulations of the W675A variant. The formation of α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages were detected from products of the W675A mutant. In contrast, the wild-type enzyme strictly synthesized α-(1,6) linkage on the maltose acceptor. This study examined the importance of W675 for transglycosylation, processivity, and regioselectivity of glucansucrases. Engineering glucansucrase active sites is one of the essential approaches to green tools for carbohydrate modification.

Reuteransucrase-catalytic kinetic modeling and functional characteristics for novel prebiotic gluco-oligomers

Gluco-oligomers synthesized via the reuteransucrase acceptor reaction from sucrose and maltose were found to be excellent prebiotic ingredient candidates for gastrointestinal health.

Synthesis of potential prebiotic α-glucooligosaccharides using microbial glucansucrase and their in vitro fecal fermentation

Novel α-glucooligosaccharides were synthesized by the acceptor reaction of Leuconostoc citreum SK24.002 glucansucrase with maltose and sucrose.

Synthesis of the rare disaccharide nigerose by structure-based design of a phosphorylase mutant with altered regioselectivity

A Q345F exchange of sucrose phosphorylase from Bifidobacterium adolescentis alters the regioselectivity of the acceptor reaction with glucose from kojibiose (α-1,2 linked) to nigerose (α-1,3 linked).