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A Single Point Mutation Converts GH84 O-GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence

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    A Single Point Mutation Converts GH84 O-GlcNAc Hydrolases into Phosphorylases: Experimental and Theoretical Evidence
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    • David Teze*
      David Teze
      Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads Bldg. 224, DK-2800 Kongens Lyngby, Denmark
      UFIP, CNRS, Université de Nantes, 44300 Nantes, France
      *[email protected]
      More by David Teze
      Orcidhttp://orcid.org/0000-0002-6865-6108
    • Joan Coines
      Joan Coines
      Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain
      More by Joan Coines
    • Lluís Raich
      Lluís Raich
      Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain
      More by Lluís Raich
    • Valentina Kalichuk
      Valentina Kalichuk
      UFIP, CNRS, Université de Nantes, 44300 Nantes, France
      More by Valentina Kalichuk
    • Claude Solleux
      Claude Solleux
      UFIP, CNRS, Université de Nantes, 44300 Nantes, France
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    • Charles Tellier
      Charles Tellier
      UFIP, CNRS, Université de Nantes, 44300 Nantes, France
      More by Charles Tellier
      Orcidhttp://orcid.org/0000-0002-5788-2847
    • Corinne André-Miral
      Corinne André-Miral
      UFIP, CNRS, Université de Nantes, 44300 Nantes, France
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    • Birte Svensson*
      Birte Svensson
      Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads Bldg. 224, DK-2800 Kongens Lyngby, Denmark
      *[email protected]
      More by Birte Svensson
      Orcidhttp://orcid.org/0000-0002-2993-8196
    • Carme Rovira*
      Carme Rovira
      Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) and Institut de Quı́mica Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain
      Institució Catalana de Recerca i Estudis Avançats, Passeig Lluís Companys 23, 08010 Barcelona, Spain
      *[email protected]
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      Orcidhttp://orcid.org/0000-0003-1477-5010
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2020, 142, 5, 2120–2124
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    https://doi.org/10.1021/jacs.9b09655
    Published January 9, 2020
    Copyright © 2020 American Chemical Society
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    Abstract

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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-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.

    Copyright © 2020 American Chemical Society

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    Cite this: J. Am. Chem. Soc. 2020, 142, 5, 2120–2124
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.9b09655
    Published January 9, 2020
    Copyright © 2020 American Chemical Society
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