Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Structural and Computational Biology Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Acta Crystallogr D Struct Biol. 2021 Aug 1;77(Pt 8):1064-1076. doi: 10.1107/S205979832100677X. Epub 2021 Jul 29.
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.
α-葡萄糖苷酶（EC 3.2.1.20）是一种碳水化合物水解酶，通常从非还原端切割寡糖和淀粉的α-1,4-糖苷键。在这项研究中，从 Weissella cibaria BBK-1 中分离出的新型α-葡萄糖苷酶（WcAG）进行了生化和结构表征。WcAG 属于糖苷水解酶家族 13（GH13）和 neo 普鲁兰酶亚家族。它对短链寡糖从还原端的α-1,4 键表现出明显的水解活性。该酶优先水解麦芽三糖和阿卡波糖，而不能水解环状寡糖和多糖。此外，WcAG 可以水解普鲁兰水解物，并在存在麦芽糖时强烈表现出转糖苷活性。分子筛层析和 X 射线晶体结构表明，WcAG 形成同源二聚体，其中一个单体的 N 端结构域定向靠近另一个单体的催化结构域，形成底物结合槽。WcAG 与麦芽糖、麦芽三糖和阿卡波糖复合物的晶体结构揭示了一个引人注目的酶活性位点，具有可及的+2、+1 和-1 亚位点，以及位于活性位点前面的 Arg-Glu 门（Arg176-Glu296）。-2 和-3 亚位点被来自不同亚基的 N 端结构域中的 Met119 和 Asn120 阻断，导致底物偏好受到极大限制。
