Investigating structure-function relationships in family 7 cellulases by molecular simulation

Abstract

The conversion of plant biomass to fermentable sugars is a primary option for the production of biofuels. Cellulases, the enzymes that break down recalcitrant plant cellulose materials into fermentable sugars, can be utilized in the commercial production of biofuels if throughput can be increased and costs reduced. Understanding the molecular-level actions of cellulases is a significant challenge that must be overcome to improve cellulase productivity and efficiency in biofuel production. Here we use molecular simulation to probe how cellulases identify and bind to crystalline cellulose and how this may be improved. The systems studied are synergistic, highly active, and well-characterized enzymes Cellobiohydrolase I (Cel7A) and Endoglucanase-I (EG1 or Cel7B) produced by the filamentous fungi Trichoderma reesei. Both of these enzymes are multi-domain, consisting of a carbohydrate binding domain (CBM), a large catalytic domain, and a connective linker peptide. In particular, we study the addition of glycans, or sugars, to the Cel7A CBM and our results suggest that changing the sugar patterns (known as glycosylation) positively impacts binding affinity by 500-fold (up to 3.8 kcal/mol). This is potentially a new direction in protein engineering in that modifying glycosylation patterns via genetic and chemical synthesis or culture manipulation can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. We also utilize molecular simulation to highlight the functional differences in the catalytic domains of Cel7A, which breaks down cellulose chains processively, and Cel7B, which randomly cleaves glycosidic bonds on the cellulose surface. By investigating the impact of protein conformational changes and aromatic mutations in the active sites of these enzymes, we offer insights into the energetics and structure-function relationships necessary for binding. In general the results of the simulations performed provide insights that could aid in the efforts to rationally engineer cellulase enzymes with improved performance in biofuel production.