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.

α-Glucosidase Inhibitors From the Coral-Associated Fungus Aspergillus terreus

“…To investigate the binding mode of these compounds with α-glucosidase, molecular docking study was carried out by using the SYBYL 2.0 software. Due to the unavailable of crystal structure of α-glucosidase from Saccharomyces cerevisiae , the crystal structure of isomaltase (PDB ID: 3A4A) from S. cerevisiae , which is 84% similar to that of S. cerevisiae α-glucosidase, was conducted as docking model (Shen et al, 2015 ). The theoretical binding mode between 4a and the enzyme was shown in Figure 7 .…”

Function and structure of GH13_31 α‐glucosidase with high α‐(1→4)‐glucosidic linkage specificity and transglucosylation activity

“…Bsp AG13_31A showed high disaccharide specificity (Tables and ), while most AGases prefer trisaccharides to disaccharides . In HaG, showing high disaccharide specificity , steric hindrance caused by long β→α loop 4 (Leu201‐Arg246) located just above the d ‐glucose residue of G2 in subsite +1 is responsible for disaccharide specificity . The overall structure of Bsp AG13_31A displayed high similarity to HaG with an RMSD of 1.8 Å for the 546 Cα atom.…”

Molecular dynamics reveals insight into how N226P and H227Y mutations affect maltose binding in the active site of α-glucosidase II from European honeybee, Apis mellifera

“…The structure of maltose was obtained from Halomonas sp. H11 αglucosidase (PDB ID: 3WY4 [21]). Their atom types and force field parameters were assigned by the GLYCAM06j-1 force field [22].…”

“…Moreover, the crystal maltose was redocked into the active site of Halomonas sp. H11 α-glucosidase (3WY4 [21]) to validate if Autodock vina and its parameters were appropriate for our systems. We…”