Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 alpha-Glucosidases

作者：Ngiwsara Lukana; Iwai Gaku; Tagami Takayoshi; Sato Natsuko; Nakai Hiroyuki; Okuyama Masayuki; Mori Haruhide^*; Kimura Atsuo

来源：Bioscience Biotechnology and Biochemistry, 2012, 76(10): 1967-1974.

DOI：10.1271/bbb.120473

• honeybee alpha-glucosidase
• glycoside hydrolase family 13
• substrate specificity
• transglucosylation

Honeybees, Apis mellifera, possess three alpha-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-M preferred sucrose to maltose as substrate and formed only alpha-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the alpha-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) alpha-glucosidases and related enzymes.

• 出版日期2012-10