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

Abstract

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.

Bacterial α-Glucan and Branching Sucrases from GH70 Family: Discovery, Structure–Function Relationship Studies and Engineering

“…citreum NRRL B-1355, ASR from Ln. citreum ABK-1 and GTF180, its replacement with another aromatic residue resulted in a decrease of activity but the transglucosylation activity was partially maintained [ 124 , 154 , 156 ]. To note, Gsy is the only GS lacking this conserved tryptophan [ 76 ].…”

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1

“…LEaP module in AMBER18 with GLYCAM06j-1 force field and ff14SB parameters [ 58 ] was used to immerse the representative binding orientations of all systems in isomeric truncated octahedral boxes of TIP3P water molecules with the 13 Å buffer distance. The five-step minimization procedure [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ] was employed to reduce the unfavorable interaction of the system. In this study, all steps used 2,500 steps of steepest descent and 2,500 steps of conjugate gradient.…”