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

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.

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

“…For example, loop 3 of some Aspergillus endoglucanases that contain [T/S]TTQA are much longer, while those from Trichoderma, Penicillium, Thielavia, and Fusarium contain [F/M/V/S]N instead. It has been reported that Asn152 of loop 3 of ThEG3 from T. harzianum forms a hydrogen bond with substrate (25). These findings suggest that loop 3 may have varied roles in catalysis.…”

“…The docking grid size was set to 66 Å by 66 Å by 66 Å, which was placed in an appropriate cavity in the catalytic pocket. The docked complexes were analyzed to select the most reliable binding conformation, as performed in previous studies (4,25). Molecular dynamics (MD) simulations were carried out using the Amber ff99SB force field at a temperature of 338 K (328 K for complexes EG and the EGΔSTTQA mutant with ligands) for 32 ns.…”

A Mini-review on the Applications of Cellulose-Binding Domains in Lignocellulosic Material Utilizations

“…A cleft structure of the hydrophobic residues (yellow) of carbohydrate-binding module family 4. Reproduced with permission from (Prates et al 2013).…”

Crystallization of Cellobiohydrolase in the Presence of Cellulose-Degraded Cellobiose: Analysis of Intermolecular Interactions and Association Dynamics

“…There have been many crystallization and structure analysis studies both for endoglucanases [7][8][9][10][11] and cellobiohydrolases [12][13][14][15][16][17] purified by several sources. These studies mainly focus on the structure of the catalytic core domain.…”