A Single Glycosidase Harnesses Different Pyranoside Ring Transition State Conformations for Hydrolysis of Mannosides and Glucosides

Hydrolysis of β-d-mannosides by β-mannosidases typically proceeds via a <i>B</i>_2,5 transition state conformation for the pyranoside ring, while that of β-d-glucosides by β-glucosidases proceeds through a distinct ⁴<i>H</i>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 <i>K</i>_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<i>E</i> conformation, while mannoimidazole bound in a <i>B</i>_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<i>S</i>₃/⁴<i>E</i> conformation in the Michaelis complex, while 4-nitrophenyl β-d-mannoside adopts a ¹<i>S</i>₅/<i>B</i>_2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of ¹<i>S</i>₃ → ⁴<i>E</i>/⁴<i>H</i>₃^⧧ → ⁴<i>C</i>₁ for β-d-glucosides and ¹<i>S</i>₅ → <i>B</i>_2,5^⧧ → ^O<i>S</i>₂ for β-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations