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The retaining β-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step

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Listed:
  • Taylor J. B. Forrester

    (University of Guelph)

  • Olga G. Ovchinnikova

    (University of Guelph)

  • Zhixiong Li

    (University of Alberta)

  • Elena N. Kitova

    (University of Alberta)

  • Jeremy T. Nothof

    (University of Alberta)

  • Akihiko Koizumi

    (University of Alberta)

  • John S. Klassen

    (University of Alberta)

  • Todd L. Lowary

    (University of Alberta
    Institute of Biological Chemistry, Academia Sinica
    National Taiwan University)

  • Chris Whitfield

    (University of Guelph)

  • Matthew S. Kimber

    (University of Guelph)

Abstract

WbbB, a lipopolysaccharide O-antigen synthesis enzyme from Raoultella terrigena, contains an N-terminal glycosyltransferase domain with a highly modified architecture that adds a terminal β-Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue to the O-antigen saccharide, with retention of stereochemistry. We show, using mass spectrometry, that WbbB forms a covalent adduct between the catalytic nucleophile, Asp232, and Kdo. We also determine X-ray structures for the CMP-β-Kdo donor complex, for Kdo-adducts with D232N and D232C WbbB variants, for a synthetic disaccharide acceptor complex, and for a ternary complex with both a Kdo-adduct and the acceptor. Together, these structures show that the enzyme-linked Asp232-Kdo adduct rotates to reposition the Kdo into a second sub-site, which then transfers Kdo to the acceptor. Retaining glycosyltransferases were thought to use only the front-side SNi substitution mechanism; here we show that retaining glycosyltransferases can also potentially use double-displacement mechanisms, but incorporating an additional catalytic subsite requires rearrangement of the protein’s architecture.

Suggested Citation

  • Taylor J. B. Forrester & Olga G. Ovchinnikova & Zhixiong Li & Elena N. Kitova & Jeremy T. Nothof & Akihiko Koizumi & John S. Klassen & Todd L. Lowary & Chris Whitfield & Matthew S. Kimber, 2022. "The retaining β-Kdo glycosyltransferase WbbB uses a double-displacement mechanism with an intermediate adduct rearrangement step," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-33988-1
    DOI: 10.1038/s41467-022-33988-1
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    References listed on IDEAS

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    1. David J. Vocadlo & Gideon J. Davies & Roger Laine & Stephen G. Withers, 2001. "Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate," Nature, Nature, vol. 412(6849), pages 835-838, August.
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