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

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 and Matthew S. Kimber ()
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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
Chris Whitfield: University of Guelph
Matthew S. Kimber: University of Guelph

Nature Communications, 2022, vol. 13, issue 1, 1-13

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-33988-1

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