Unmasking the Mystery Base Employed By the T. Reesei Cel6A Cellulase

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.