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Hydrolysis of β-d-mannosides by β-mannosidases typically proceeds via a B 2,5 transition state conformation for the pyranoside ring, while that of β-d-glucosides by β-glucosidases proceeds through a distinct 4 H 3 transition state conformation. However, rice Os7BGlu26 β-glycosidase hydrolyzes 4-nitrophenyl β-d-glucoside and β-d-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGlu26 with K i values of 2.7 nM and 10.4 μM, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a 4 E conformation, while mannoimidazole bound in a B 2,5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl β-d-glucoside adopts a 1 S 3/4 E conformation in the Michaelis complex, while 4-nitrophenyl β-d-mannoside adopts a 1 S 5/B 2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of 1 S 3 → 4 E/4 H 3 ⧧ → 4 C 1 for β-d-glucosides and 1 S 5 → B 2,5 ⧧ → O S 2 for β-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.

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