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.
Highlights
Carbohydrates are an essential biomolecule which play important roles in biological functions
The results showed that wild-type ALT (WT) could effectively control the orientation of maltose bound in the active site; O6 of the nonreducing end of maltose was close enough and in an appropriate orientation to attack C1 of the glc-D635 intermediate and form a panose molecule with α-(1, 6) linkage (Figure 7A)
W675 provided hydrophobic stacking to maltose, but it formed hydrogen bonds with maltose in WT. It did not form such bonds in the W675A mutant. These results indicated the importance of W675 in controlling the orientation of maltose binding in the active site of alternansucrase
Summary
Carbohydrates are an essential biomolecule which play important roles in biological functions (e.g., cell structure, cell recognition, and as energy sources). They are compositional parts of other biomolecules. The study of carbohydrate structures is very difficult due to the high diversity of monosaccharides, the substitution of OH groups, and myriad linkage types. [2,3] To overcome these issues, enzymatic synthesis of carbohydrates could be developed, in order to take advantage of the high specificity and turnover rate. Oligosaccharides and glucans synthesized from glucansucrases are economical in cost; their sole substrate is sucrose, or table sugar
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