Biochemical characterization of general acid/base glutamate mutants E236Q/S of dextran glucosidase and structural insights into trisaccharide-specificity and O-glycoligase activity
摘要
Streptococcus mutans dextran glucosidase (SmDG) hydrolyzes α-(1 → 6)-d-glucosides, including isomaltooligosaccharides and panose. This enzyme belongs to glycoside hydrolase family 13 (GH13) in sequence-based classification. Previous structural analysis suggests that Glu236 of SmDG is a general acid/base catalyst, but its function is not conclusively established. Additionally, our understanding of the substrate-binding mechanism of SmDG is limited, as only the structure of the complex with isomaltotriose was analyzed. Herein, we biochemically investigated the function of Glu236 and elucidated the structures of SmDG complexes with isomaltose and panose. The E236Q mutation caused a greater reduction in kcat/Km for isomaltose than for p-nitrophenyl α-d-glucoside (pNPG) and α-d-glucosyl fluoride (GF), both of which harbor good leaving groups. NaN3 supplementation increased the reaction rate of SmDG E236Q for pNPG, and α-d-glucosyl azide was produced. These results provide functional evidence for Glu236 as the general acid/base catalyst. Crystal structures of SmDG E236Q in complex with isomaltose and panose were determined at 2.30 and 2.60 Å, respectively. The Trp238 side chain of SmDG E236Q adopted a similar orientation in complexes with isomaltotriose and panose; however, it more extensively covered the d-glucose residue of panose at subsite + 2 than that of isomaltotriose, consistent with the higher preference for panose. In contrast, the Trp238 side chain did not participate in isomaltose binding and instead rotated away from the active site. SmDG E236S effectively catalyzed O-glycoligase reactions, in which d-glucosyl residue is transferred from GF to maltose, kojibiose, and nigerose to form α-(1 → 6)-d-glucosidic linkages, whereas SmDG E236Q did not exhibit such activity.