INTERACTION OF CEL9A WITH CELLULOSE FIBERS BY COMPUTER SIMULATIONS

FQ08

Osmair Vital de Oliveira (osmair07@hotmail.com) (1,2), Dr. Luiz Carlos Gomide Freitas (2), Tjerk P. Straatsma (1) and Roberto Dias Lins (1)

(1) Pacific Northwest National Laboratory, Richland, WA, USA
(2) Departamento de Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil

Biofuels are sought as an alternative to reduce the world’s dependence on non-renewable resources (petroleum, natural gas, coal). The most common fuel nowadays is ethanol, currently extracted from the corn grain (starch) and sugar cane (sucrose). The breakdown to sugars from cellulosic biomass constitutes a logical and environmental sustainable option of energy source. Plant cell-wall degrading enzymes, cellulases, are produced by a variety of fungi and bacteria. However, the use of cellulases to convert cellulosic biomass into liquid fuel involves the cost-effective production of efficient enzymes.  
 
Glycoside hydrolases process polysaccharides relatively inefficiently. The rate-limiting step in hydrolysis is not the catalytic cleavage, but the disruption of a single chain from its native matrix, which is often inaccessible to the active site of the appropriate enzymes. For this reason, enzymes in aqueous solutions have difficulty to act on the insoluble, highly ordered cellulose matrix. The interaction of the cellulases with the cellulose matrix is however mediated by a single scaffoldin-borne cellulose-binding module (CBM), overcoming the problem of cellulose binding. In fact, an enhancement in the catalytic activity for several cellulases has been observed upon attachment of CBMs. It has also been reported that the deletion of the CBM from the family IIIc rendered the enzyme almost completely inactive. Therefore, the understanding of the molecular level interactions between protein and substrate is essential for the design of enzymes with high catalytic efficiency.  
 
In order to characterize such interactions, molecular docking and molecular dynamics simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. This bacterium synthesizes a total of six cellulases: three endocellulases, two exocellulases and Cel9A, which acts as endo- and exocellulase. Among those, Cel9A is responsible for a higher soluble oligosaccharide output from insoluble cellulose and was the first enzyme identified to comprise both domains, catalytic and CBM. The dual characteristic of this enzyme makes it an ideal model since it allows for the simultaneous probing of carbohydrate recognition/attachment and catalytic activity.
 
The simulations show that the fiber binds to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain with a polysaccharide chain. The results also expand the previously known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 residues were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.