Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116

Abstract

Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on Thermoanaerobacterium xylanolyticum TxGH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in KM for p-nitrophenyl-β-D-glucoside, and significant decreases in the kcat, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the kcat/KM by over 60,000-fold, while R786 mutants increased both the KM (40-fold) and kcat (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol−1, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications.