title: Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism.
abstract: α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long β → α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure.
authors.affiliation: School of Light Industry and Food Science, South China University of Technology, 381 Wushan Road, Tianhe District, Guangzhou, Guangdong 510640, People's Republic of China.
authors.forename: Xing
authors.initials: X
authors.lastname: Shen
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8689, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Saburi
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Zuoqi
authors.initials: Z
authors.lastname: Gai
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kato
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Teruyo
authors.initials: T
authors.lastname: Ojima-Kato
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Jian
authors.initials: J
authors.lastname: Yu
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Komoda
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8689, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kido
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8689, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Matsui
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8689, Japan.
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003460:Crystallization; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000520:alpha-Glucosidases
keywords: Halomonas sp. H11; glucosyl-enzyme intermediate; glycoside hydrolase family 13; reaction mechanism; substrate specificity; α-glucosidase