Quantum mechanical/molecular mechanical molecular dynamics simulation of wild-type and seven mutants of CpNagJ in complex with PUGNAc.

The enzyme O-glycoprotein 2-acetamino-2-deoxy-beta-d-glucopyranosidase (O-GlcNAcase) is responsible for the removal of N-acetylglucosamine moieties from 2-acetamido-2-deoxy-beta-D-glucopyranose (O-GlcNAc) residues of serine/threonine residues of modified proteins. We herein present results of hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations applied to the study of the interactions established between a bacterial Clostridium perfringens homologue (CpNagJ) and PUGNAc, a potent known inhibitor of this enzyme. Electrostatic binding free energy and energy term decomposition have been computed for the wild-type CpNagJ and several mutants: D297N, D298N, Y335F, N390A, N396A, D401A, and W490A. The theoretical results have been compared with recently experimental data based on crystallographic and mutation studies on the same system. Our results reveal that, first, there is a strong interaction between Asp401, Asp298, and Asp297 residues and the PUGNAc inhibitor; and, second, the electrostatic substrate binding free energy is higher in wild-type, N390A and W490A mutants than in D297N, D298N, Y335F, N396A, and D401A ones, in accordance with the experimental results. Finally, both our theoretical predictions and the experimental data are compatible with a substrate-assisted reaction mechanism, involving two conserved aspartate residues.