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Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on β→α Loop 5

Authors :
Waraporn Auiewiriyanukul
Wataru Saburi
Tomoya Ota
Jian Yu
Koji Kato
Min Yao
Haruhide Mori
Source :
Molecules, Vol 28, Iss 7, p 3109 (2023)
Publication Year :
2023
Publisher :
MDPI AG, 2023.

Abstract

α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.

Details

Language :
English
ISSN :
14203049
Volume :
28
Issue :
7
Database :
Directory of Open Access Journals
Journal :
Molecules
Publication Type :
Academic Journal
Accession number :
edsdoj.bea95258819c4095830ee6b2c5134d02
Document Type :
article
Full Text :
https://doi.org/10.3390/molecules28073109