(557e) Breaking Down Cellulose – the Catalytic Mechanism of a Key Cellulase Elucidated By Path Sampling

The enormous reserves of carbohydrates found in plant cell walls represent a promising potential source of renewable energy.  Deconstruction of these molecules, either thermochemically or biochemically, precedes the ability to transform them to commodity chemicals or fuels.  The biochemical route involves the utilization of enzyme cocktails that synergistically break down the biopolymers.  Glycoside hydrolases (GHs) are responsible for myriad transformations of carbohydrate glycosidic linkages including polysaccharide turnover in the biosphere. GHs typically hydrolyze glycosidic bonds via mechanisms that either invert or retain the stereochemistry of the anomeric carbon. Retaining mechanisms consist of two elementary steps that include the formation of an enzyme-glycosyl intermediate and a deglycosylation step wherein a water molecule attacks the anomeric carbon to complete the catalytic cycle. Here we examine the two-step retaining mechanism in the Hypocrea jecorina GH Family 7 cellobiohydrolase (Cel7A), a key cellulose-degrading enzyme.  Cel7A is often a major component in industrial cellulase cocktails and the target of cellulase engineering studies. We utilize transition path sampling with mixed quantum-mechanics/molecular mechanics (QM/MM) simulations to harvest many trajectories for the glycosylation and deglycosylation steps. Subsequent analysis of these trajectories via Likelihood Maximization yields the reaction coordinate for each step, which is verified using the histogram test. The reaction coordinates involve forming/breaking bonds, as well as less-intuitive elements such as catalytic residue orientation and possible product (i.e. cellobiose) assistance. From there, we compute the full free energy surface for the two-step mechanism, including the movement of the cellobiose product from the product of the glycosylation step mode to the conformation that occurs in between the two catalytic reactions. Additionally, we quantify the puckering of the -1 glucosyl residue along the reaction pathway. Overall, this study provides direct insight into the full catalytic mechanism for a two-step, retaining cellobiohydrolase.