Advantages of a distant cellulase catalytic base

Tucker Burgin,Jerry Ståhlberg,Heather B Mayes

doi:10.1074/jbc.ra117.001186

Abstract

The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels. Until recently, its catalytic mechanism had been uncertain: The best candidate residue to serve as a catalytic base, Asp-175, is farther from the glycosidic cleavage site than in other glycoside hydrolase enzymes. Recent unbiased transition path sampling simulations revealed the hydrolytic mechanism for this more distant base, employing a water wire; however, it is not clear why the enzyme employs a more distant catalytic base, a highly conserved feature among homologs across different kingdoms. In this work, we describe molecular dynamics simulations designed to uncover how a base with a longer side chain, as in a D175E mutant, affects procession and active site alignment in the Michaelis complex. We show that the hydrogen bond network is tuned to the shorter aspartate side chain, and that a longer glutamate side chain inhibits procession as well as being less likely to adopt a catalytically productive conformation. Furthermore, we draw comparisons between the active site in Trichoderma reesei Cel6A and another inverting, processive cellulase to deduce the contribution of the water wire to the overall enzyme function, revealing that the more distant catalytic base enhances product release. Our results can inform efforts in the study and design of enzymes by demonstrating how counterintuitive sacrifices in chemical reactivity can have worthwhile benefits for other steps in the catalytic cycle.

Highlights

The inverting glycoside hydrolase Trichoderma reesei (Hypocrea jecorina) Cel6A is a promising candidate for protein engineering for more economical production of biofuels
Koivula et al [15] proposed that the two water molecules that consistently appear in the active site of crystal structures would act as a water wire to shuttle a proton to Trichoderma reesei Cel6A (TrCel6A) Asp-175, allowing this residue to serve as the catalytic base
To bridge the gap between atomistic enzymatic detail and human chemical intuition, we constructed molecular models of both wildtype and D175E TrCel6A and investigated the influence of the mutation on two nonreactive steps in the catalytic cycle: 1) procession of the substrate into the active site and 2) the transition to the reaction-competent active site conformation following procession. We found in both cases that the longer Glu-175 residue in the mutant was a hindrance to the catalytic cycle, highlighting the importance and intricacy of the remarkable network of hydrogen bonding interactions that stabilizes the active site of the wildtype enzyme

Summary

Advantages of a distant cellulase catalytic base

To bridge the gap between atomistic enzymatic detail and human chemical intuition, we constructed molecular models of both wildtype and D175E TrCel6A and investigated the influence of the mutation on two nonreactive steps in the catalytic cycle: 1) procession of the substrate into the active site and 2) the transition to the reaction-competent active site conformation following procession We found in both cases that the longer Glu-175 residue in the mutant was a hindrance to the catalytic cycle, highlighting the importance and intricacy of the remarkable network of hydrogen bonding interactions that stabilizes the active site of the wildtype enzyme. Based on the results of these studies, we propose a benefit in cellulases to activation of the nucleophilic water via a wire water by taking into account aspects of the enzymatic cycle outside the reaction itself, broadening the context for rationalizing enzymatic features in carbohydrate-active enzymes

Results

Active site homology

Discussion

Molecular dynamics simulations