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

Author(s)

Prates, Erica T. ^[1] ; Stankovic, Ivana ^[1] ; Silveira, Rodrigo L. ^[1] ; Liberato, Marcelo V. ^[2] ; Henrique-Silva, Flavio ^[3] ; Pereira, Jr., Nei ^[4] ; Polikarpov, Igor ^[2] ; Skaf, Munir S. ^[1]

Affiliation

^[1] State Univ Campinas UNICAMP, Inst Chem, Campinas, SP - Brazil

^[2] Univ Sao Paulo, Inst Phys Sao Carlos, Sao Carlos, SP - Brazil

^[3] Univ Fed Sao Carlos, Dept Genet & Evolucao, BR-13560 Sao Carlos, SP - Brazil

^[4] Univ Fed Rio de Janeiro, Ctr Tecnol, Escola Quim, Lab Desenvolvimento Bioproc LaDeBio, Rio De Janeiro - Brazil

• Document type: Journal article
• Source: PLoS One; v. 8, n. 3 MAR 14 2013.
• Web of Science Citations: 18

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. (AU)

• FAPESP's process: 10/08680-2 - Molecular aspectos of lignocellulosic biomass degradation: dynamics of enzymes and plant cell wall nanoarchitecture
• Grantee: Rodrigo Leandro Silveira
• Support type: Scholarships in Brazil - Doctorate
• FAPESP's process: 09/54035-4 - Facility for advanced studies of biosystems and nanostructured materials
• Grantee: Igor Polikarpov
• Support type: Multi-user Equipment Program
• FAPESP's process: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
• Grantee: Igor Polikarpov
• Support type: Program for Research on Bioenergy (BIOEN) - Thematic Grants