10-06: Understanding fungal cellulases with molecular simulation

Fungal cellulases form the basis of many industrial enzyme cocktails for biomass conversion to fuels. Here, we describe a large set of simulation studies, with accompanying experiments where possible, aimed at understanding how the cellulases, Cel7A, Cel7B, and Cel6A from the fungus Trichoderma reesei, deconstruct cellulose to cellobiose. Regarding the carbohydrate-binding modules (CBMs), we demonstrate that O-glycosylation has a significant impact on the CBM binding affinity, which may have ramifications for glycoprotein engineering of cellulases since glycans vary as a function of expression host and growth conditions. With simulation, we predict that product inhibition varies dramatically between processive enzymes and non-processive enzymes, such that distinct populations of these enzymes must be accounted for when measuring product inhibition. From mutational simulations, we examine the aromatic-carbohydrate interactions in the Cel7A and Cel6A catalytic domain tunnels and demonstrate that these interactions vary widely depending on the protein fold and the location within the tunnel. Additionally, we demonstrate with simulation that pH exhibits a significant effect on the structure of the catalytic domains of fungal cellulases, which may have significant ramifications for complexation and catalysis. Lastly, we conducted long molecular dynamics simulations of the Cel7A and Cel6A enzymes on cellulose, and predict that the linker may possess more function previously considered, and that several conserved loops on the catalytic domains interact with and aid in cellulose deconstruction. Overall, these results provide new insights into our collective understanding of the mechanisms by which fungal cellulases deconstruct cellulose.