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 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.

Back to (non-)Basics: An Update on Neutral and Charge-Balanced Glycosidase Inhibitors

“…158,159 This is supported by a broader understanding of the tertiary structures of enzyme targets and the further uncovering of novel mechanisms in this family. 98,160,161 Epoxides such as the natural product cyclophellitol and sulfonium ions like ones found naturally within the salicinol family were both discovered as synthetic classes of glycosidase inhibitor before they were identified in nature. 84,146 The irreversible nature of epoxide inhibitors has recently allowed detection of proteins fused to a bglucosidase in a cellular context.…”

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase

“…The same procedure has been previously used to analyze the conformational preferences of related GH inhibitors (mannoimidazole, glucoimidazole, and IFG). 39 , 40 …”

Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

“…In fact, the investigation of TS conformation is useful for predicting conformational itineraries (from reactant to product state via the TS) based on the principle that the reaction pathway with the least nuclear motion, i.e., the least change in atomic position and electronic configuration will be favored [ 28 ]. Glucoimidazole (GIm) and mannoimidazole (MIm) inhibitors ( Figure 1 B,C) are qualitatively good models of a pyranosyl oxocarbenium ion-like TS for rice Os7BGlu26 β-mannosidase and similarly showed inhibition of Os3BGlu7 [ 29 ]. Os3BGlu7 had 4420-fold higher affinity (lower competitive inhibition constant) for GIm than for MIm, corresponding to a ~5 kcal/mol difference in binding free energy [ 29 ].…”

“…Glucoimidazole (GIm) and mannoimidazole (MIm) inhibitors ( Figure 1 B,C) are qualitatively good models of a pyranosyl oxocarbenium ion-like TS for rice Os7BGlu26 β-mannosidase and similarly showed inhibition of Os3BGlu7 [ 29 ]. Os3BGlu7 had 4420-fold higher affinity (lower competitive inhibition constant) for GIm than for MIm, corresponding to a ~5 kcal/mol difference in binding free energy [ 29 ]. However, Os7BGlu26 β-mannosidase also showed ~5 kcal/mol more favorable binding energy for GIm than MIm, despite binding MIm in a TS-like conformation and showing a preference for mannoside over glucoside for hydrolysis.…”