title: Amino acids in conserved region II are crucial to substrate specificity, reaction velocity, and regioselectivity in the transglucosylation of honeybee GH-13 α-glucosidases.
abstract: Honeybees, Apis mellifera, possess three α-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-III preferred sucrose to maltose as substrate and formed only α-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the α-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) α-glucosidases and related enzymes.
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
authors.forename: Lukana
authors.initials: L
authors.lastname: Ngiwsara
authors.forename: Gaku
authors.initials: G
authors.lastname: Iwai
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Tagami
authors.forename: Natsuko
authors.initials: N
authors.lastname: Sato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D001516:Bees / Q000201:enzymology*; D017124:Conserved Sequence; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013237:Stereoisomerism; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000520:alpha-Glucosidases