A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides

Abstract

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and −1 subsites, along with an Arg–Glu gate (Arg176–Glu296) in front of the active site. The −2 and −3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

Novel Insights Into the Phylogeny and Biotechnological Potential of Weissella Species

“…The substrates for this hydrolytic enzyme are maltooligosaccharides, phenyl α-maltoside, nigerose, soluble starch, amylose, amylopectin, and β-limit dextrins (Tomasik and Horton, 2012 ). In a recent study by Wangpaiboon et al ( 2021 ), this enzyme was characterized in W. cibaria as acting on short-chain maltooligosaccharides. Genes encoding 6-phospho-β-glucosidase enzymes were annotated in the W. diestrammenae, W. fabalis, W. fabaria , and W. ghanensis type strains.…”

Comparative genome analysis of four Leuconostoc strains with a focus on carbohydrate-active enzymes and oligosaccharide utilization pathways

“…can utilize specific oligosaccharides. GH13_30 is also found in all Leuconostoc strains and is classified as α-glucosidase (EC 3.2.1.20), a carbohydrate-hydrolyzing enzyme that hydrolyzes maltotriose by breaking α-1,4-glycosidic bonds [61] . GH13_31 and GH65, annotated as 1,6-α-glucosidase (EC 3.2.1.10, also known as isomaltose and alpha-methyl glucosidases) and maltose phosphorylase (EC 2.4.1.8), respectively, are present in all Leuconostoc spp.…”

Structure and Function Insight of the α-Glucosidase QsGH13 From Qipengyuania seohaensis sp. SW-135

“…The loop-rich domain B (residues 108–175) is derived from the domain A, including an α-helix and three β-fold, which are involved in the formation of QsGH13 dimers and forms catalytic pockets with domain A. Domain C (residues 464–525) is composed of multiple highly conserved β-folds that stabilizes the entire protein structure ( Shen et al, 2015 ; Figures 3B,C ). In order to achieve catalytic activity, dimers formed by two monomers are essential in the GH13 family of enzymes ( Watanabe et al, 2020 ; Wangpaiboon et al, 2021 ). The QsGH13 monomers formed dimers mainly through five hydrogen bonds and 105 non-bonded contacts ( Supplementary Figure 4 ).…”