A single glycosidase harnesses different pyranoside ring transition state conformations for hydrolysis of mannosides and glucosides

Abstract

Hydrolysis of β-D-mannosides by β-mannosidases typically proceeds via a B₂‚₅ transition state conformation for the pyranoside ring, while that of β-D-glucosides by β-glucosidases proceeds through a distinct ⁴H₃ 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 Ki 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 ⁴E conformation, while mannoimidazole bound in a B₂,₅ 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 ¹S3/4E conformation in the Michaelis complex, while 4-nitrophenyl β-D-mannoside adopts a ¹S₅⁄B₂,₅ conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of ¹S₃ → ⁴E/⁴H₃ ⧧ → ⁴C₁ for β-D-glucosides and ¹S₅ → B₂,₅ ⧧ → ⁰S₂ for β-D-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.