Molecular mechanisms of cellulose processivity and hydrolysis via advanced molecular simulations

Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event. Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we employ advanced computational tools to study key steps in the processive cycle of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A. Namely, we examine cellooligomer chain threading through the binding tunnel, catalytic activation, and both chemical steps (i.e. glycosylation and deglycosylation). The first chemical step, glycosylation, cleaves cellobiose from the cellulose chain and forms a glycosyl-enzyme intermediate (GEI). Deglycosylation follows, wherein a nucleophilic water molecule breaks the GEI and restores the catalytic residues. We calculate free energy barriers for both the processive and the hydrolytic steps, as well as employing transition path sampling to accurately determine the reaction mechanism for the slower chemical steps. In addition to revealing key details of the processive cycle of this naturally and industrially important enzyme family, we present a general computational framework that contributes to the development of structure-function relationships and yields kinetically meaningful insight into enzymatic action.