Mechanism of a Hotdog-Fold Acyl-CoA Thioesterase By QM/MM Metadynamics Simulation

Many thioesterases have a structural HotDog fold, while others have alpha/beta folds. Based on crystal structures, putative mechanisms have been proposed for some HotDog-fold thioesterases. The reaction of a human thioesterase enzyme (hTHEM2), part of thioesterase family 8 in the ThYme database and one of 25 current families, was explored by first-principles methods (Car-Parrinello molecular dynamics) to elucidate atomic and electronic details of the mechanism, its transition-state conformation, and the energy of the process. Mixed quantum mechanics/molecular mechanics simulations using the metadynamics technique were used, and an acid-base-like mechanism was found. The activation of nucleophilic water by an aspartate residue acting as a base, as well as a tetrahedral-like intermediate, were confirmed as previously proposed. Additionally, new evidence pointing toward thioester substrate protonation from a histidine residue via a serine residue is presented. To our knowledge, this is the first time a thioesterase with any fold has been studied by first-principles methods.