QM/MM studies on the glycosylation mechanism of rice BGlu1 β-glucosidase.

The quantum-mechanical/molecular-mechanical (QM/MM) method was used to study the glycosylation mechanism of rice BGlu1 β-glucosidase in complex with laminaribiose. The calculation results reveal that the glycosylation step experiences a concerted process from the reactant to the glycosyl-enzyme complex with an activation barrier of 15.7 kcal/mol, in which an oxocarbenium cation-like transition state (TS) is formed. At the TS, the terminal saccharide residue planarizes toward the half-chair conformation, and the glycosidic bond cleavage is promoted by the attacks of proton donor (E176) on glycosidic oxygen and nucleophilic residue (E386) on the anomeric carbon of laminaribiose. Both the nucleophilic glutamate (E386) and acid/base catalyst (E176) establish shorter hydrogen bridges with the C₂-hydroxyl groups of sugar ring, which play an important role in the catalytic reaction of rice BGlu1 β-glucosidase.