(484h) Unmasking the Mystery Base Employed By the T. Reesei Cel6A Cellulase

Mayes, H. B., Northwestern University
Knott, B. C., National Renewable Energy Laboratory
Crowley, M. F., National Renewable Energy Laboratory
Götz, A. W., San Diego Supercomputer Center
Broadbelt, L. J., Northwestern University
Beckham, G. T., National Renewable Energy Laboratory

Trichoderma reesei Cel6A is an industrially-important enzyme for converting cellulose to renewable fuels and chemicals. Still, many questions remain for the understanding of its catalytic mechanism despite many structural and kinetic studies with both wild-type and mutant enzymes. Cel6A is a cellobiohydrolase (CBH) that binds the cellulose chain in a tunnel enclosed by extended loops, which enables processive action where multiple cellobiose units are cleaved off from the non-reducing end of the chain before enzyme detachment. The glycosidic bond is hydrolyzed by a single-step inverting reaction mechanism, i.e. single displacement nucleophilic substitution at the anomeric carbon by an activated water molecule. However, the identity of the Cel6A catalytic base that abstracts a proton to activate the water molecule has remained a mystery. Path-sampling offers the tools for uncovering the reactive potential energy surface of the catalytic event, thus providing a molecular-level understanding of the roles of each residue in the cleaving of the strong glycosidic bond. Our combined quantum mechanics and molecular mechanics (QM/MM) simulations reveal the key role of a water wire in the shuttling of a proton away from the active site to the putative base, as well as providing a means for catalytic rescue upon mutation of the putative base. In addition to providing an atomistic understanding of enzyme action, this model provides a tool for the rational design of more efficient enzymes for use in producing renewable chemicals and fuels from non-food biomass.