Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 142, Issue 5, Pages 2120-2124Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.9b09655
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Funding
- MICINN [CTQ2017-85496-P, MDM-2017-0767]
- AGAUR [2017SGR-1189]
- Carlsberg Foundation
- Villum Foundation (DTU NMR Center)
- NNF [NNF170C0025660]
- MINECO [FPI-BES-2015-072055]
- BSC-CNS [QSB-2019-3-0001]
- DFF
- DFT
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Glycoside hydrolases and phosphorylases are two major classes of enzymes responsible for the cleavage of glycosidic bonds. Here we show that two GH84 O-G1cNAcase 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.
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