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

Autor: Forrester TJB; Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road E., Guelph, ON, N1G 2W1, Canada., Ovchinnikova OG; Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road E., Guelph, ON, N1G 2W1, Canada., Li Z; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada., Kitova EN; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada., Nothof JT; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada., Koizumi A; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada., Klassen JS; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada., Lowary TL; Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, AB, T6G 2G2, Canada.; Institute of Biological Chemistry, Academia Sinica, Academia Road, Section 2, #128, Nangang, Taipei, 11529, Taiwan.; Institute of Biochemical Sciences, National Taiwan University, Section 4, #1, Roosevelt Road, Taipei, 10617, Taiwan., Whitfield C; Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road E., Guelph, ON, N1G 2W1, Canada., Kimber MS; Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road E., Guelph, ON, N1G 2W1, Canada. mkimber@uoguelph.ca.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2022 Oct 21; Vol. 13 (1), pp. 6277. Date of Electronic Publication: 2022 Oct 21.
DOI: 10.1038/s41467-022-33988-1
Abstrakt: 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 S N i 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.
(© 2022. The Author(s).)
Databáze: MEDLINE
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