Abstract

β-Glucosidases and β-mannosidases hydrolyze substrates that differ only in the epimer of the nonreducing terminal sugar moiety, but most such enzymes show a strong preference for one activity or the other. Rice Os3BGlu7 and Os7BGlu26 β-glycosidases show a less strong preference, but Os3BGlu7 and Os7BGlu26 prefer glucosides and mannosides, respectively. Previous studies of crystal structures with glucoimidazole (GIm) and mannoimidazole (MIm) complexes and metadynamic simulations suggested that Os7BGlu26 hydrolyzes mannosides via the B2,5 transition state (TS) conformation preferred for mannosides and glucosides via their preferred 4H3/4E TS conformation. However, MIm is weakly bound by both enzymes. In the present study, we found that MIm was not bound in the active site of crystallized Os3BGlu7, but GIm was tightly bound in the −1 subsite in a 4H3/4E conformation via hydrogen bonds with the surrounding residues. One-microsecond molecular dynamics simulations showed that GIm was stably bound in the Os3BGlu7 active site and the glycone-binding site with little distortion. In contrast, MIm initialized in the B2,5 conformation rapidly relaxed to a E3/4H3 conformation and moved out into a position in the entrance of the active site, where it bound more stably despite making fewer interactions. The lack of MIm binding in the glycone site in protein crystals and simulations implies that the energy required to distort MIm to the B2,5 conformation for optimal active site residue interactions is sufficient to offset the energy of those interactions in Os3BGlu7. This balance between distortion and binding energy may also provide a rationale for glucosidase versus mannosidase specificity in plant β-glycosidases.

Highlights

  • The largest source of renewable biomass on earth is plant-derived carbohydrate, including cellulose and other β-glucans, as well as xylans, mannans and mixed polysaccharides

  • We solved the structure of Os7BGlu26 β-mannosidase in complex with both GIm and MIm, each of which was found in the shape expected for their respective sugar transition state (TS) conformation of 4H3/4E and B2,5 [29]

  • We have shown that GIm binds tightly in the −1 subsite of Os3BGlu7 in its relaxed 4H3/4E conformation, corresponding to the TS structure for β-glucosidases

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Summary

Introduction

The largest source of renewable biomass on earth is plant-derived carbohydrate, including cellulose and other β-glucans, as well as xylans, mannans and mixed polysaccharides. Most β-glucosidases are thought to catalyze hydrolysis through a two-step retaining mechanism involving two catalytic carboxylate residues (two glutamate residues for GH1 enzymes), one of which serves as a general acid/base catalyst and another as a nucleophile [12,13]. A water molecule or another nucleophile is deprotonated by the catalytic acid/base and attacks the anomeric carbon to release β-d-glucose from the enzyme Both steps are thought to proceed via the formation of oxocarbenium cation-like transitions states (TS). Os7BGlu β-mannosidase 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 Biomolecules 2020, 10, 907 mannoside over glucoside for hydrolysis. The final models were analyzed with PROCHECK [34] and validated on the PDB website

System Preparation for Molecular Modeling
Molecular Dynamics Simulations
Replica Exchange Molecular Dynamics of the Unbound Glycoside Inhibitors
Structure of Rice Os3BGlu7 in Complex with Inhibitor
Inhibitor Binding Pattern
Ligand-Binding Pocket Volume and Water Accessibility in the Binding Pocket
Conclusions
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