X-ray structure and molecular dynamics simulations of endoglucanase 3 from Trichoderma harzianum: structural organization and substrate recognition by endoglucanases that lack cellulose binding module.

Munir S. Skaf,Érica T. Prates,Rodrigo L. Silveira,Nei Pereira,Igor Polikarpov,Marcelo V. Liberato,Flávio Henrique-Silva,Chandra Verma,Ivana Stankovic

doi:10.1371/journal.pone.0059069

Abstract

Plant biomass holds a promise for the production of second-generation ethanol via enzymatic hydrolysis, but its utilization as a biofuel resource is currently limited to a large extent by the cost and low efficiency of the cellulolytic enzymes. Considerable efforts have been dedicated to elucidate the mechanisms of the enzymatic process. It is well known that most cellulases possess a catalytic core domain and a carbohydrate binding module (CBM), without which the enzymatic activity can be drastically reduced. However, Cel12A members of the glycosyl hydrolases family 12 (GHF12) do not bear a CBM and yet are able to hydrolyze amorphous cellulose quite efficiently. Here, we use X-ray crystallography and molecular dynamics simulations to unravel the molecular basis underlying the catalytic capability of endoglucanase 3 from Trichoderma harzianum (ThEG3), a member of the GHF12 enzymes that lacks a CBM. A comparative analysis with the Cellulomonas fimi CBM identifies important residues mediating interactions of EG3s with amorphous regions of the cellulose. For instance, three aromatic residues constitute a harboring wall of hydrophobic contacts with the substrate in both ThEG3 and CfCBM structures. Moreover, residues at the entrance of the active site cleft of ThEG3 are identified, which might hydrogen bond to the substrate. We advocate that the ThEG3 residues Asn152 and Glu201 interact with the substrate similarly to the corresponding CfCBM residues Asn81 and Arg75. Altogether, these results show that CBM motifs are incorporated within the ThEG3 catalytic domain and suggest that the enzymatic efficiency is associated with the length and position of the substrate chain, being higher when the substrate interact with the aromatic residues at the entrance of the cleft and the catalytic triad. Our results provide guidelines for rational protein engineering aiming to improve interactions of GHF12 enzymes with cellulosic substrates.

Highlights

Bioethanol is an attractive renewable fuel that has been produced in large quantities by the alcoholic fermentation of concentrated syrups obtained from sugar cane, corn and other feedstocks in countries such as Brazil and the United States, aiming to supplement and, eventually, to replace fossil liquid fuels
These results show that carbohydrate binding module (CBM) motifs are incorporated within the ThEG3 catalytic domain and suggest that the enzymatic efficiency is associated with the length and position of the substrate chain, being higher when the substrate interact with the aromatic residues at the entrance of the cleft and the catalytic triad
We report the three-dimensional structure of ThEG3 obtained from X-ray crystallography and investigate enzyme-substrate interactions using molecular dynamics (MD) simulations

Summary

Introduction

Bioethanol is an attractive renewable fuel that has been produced in large quantities by the alcoholic fermentation of concentrated syrups obtained from sugar cane, corn and other feedstocks in countries such as Brazil and the United States, aiming to supplement and, eventually, to replace fossil liquid fuels. Over the last several years, increasing research efforts have been devoted to the production of the second-generation cellulosic ethanol, in which cellulosic biomass is chemically and enzymatically degraded into soluble fermentable sugars. Under industrial settings, the enzymatic catalysis is one of the most expensive steps of the biomass-to-cellulosic ethanol bioconversion process due to the low efficiency and the high cost of currently available enzyme preparates. In order to develop strategies for reducing the costs of this process, extensive efforts have been directed to the study of cellulolytic microorganisms and to investigate the mechanisms of biomass enzymatic hydrolysis, as well as general structural and dynamic properties of glycosyl hydrolases (GHs)

Objectives

Methods

Results

Conclusion