A single point mutation converts GH84 O-GlcNAc hydrolases into phosphorylases. Experimental and theoretical evidence.

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 ten-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. In addition, the simulations unambiguously reveal the nature of the intermediate as a glucose oxazolinium ion, clarifying the debate on the nature of such reaction intermediate in glycoside hydrolases operating via substrate-assisted catalysis.