Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-GlcNAcase enzymes can be converted to efficient phosphorylases by a single point mutation. Noteworthy, the mutated enzymes are over 10-fold more active than naturally occurring glucosaminide phosphorylases. We rationalize this novel transformation using molecular dynamics and QM/MM metadynamics methods, showing that the mutation changes the electrostatic potential at the active site and reduces the energy barrier for phosphorolysis by 10 kcal·mol-1. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such a reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.
Teze, D., Coines, J., Raich, L., Kalichuk, V., Solleux, C., Tellier, C., ... Rovira, C. (2020). A Single Point Mutation Converts GH84 O-GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence. Journal of the American Chemical Society, 142(5), 2120-2124. https://doi.org/10.1021/jacs.9b09655