Details

Autor(en) / Beteiligte

• Forrester, Taylor J. B.
• Ovchinnikova, Olga G.
• Li, Zhixiong
• Kitova, Elena N.
• Nothof, Jeremy T.
• Koizumi, Akihiko
• Klassen, John S.
• Lowary, Todd L.
• Whitfield, Chris
• Kimber, Matthew S.

Titel

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

Ist Teil von

• Nature communications, 2022-10, Vol.13 (1), p.6277-6277, Article 6277

Ort / Verlag

London: Nature Publishing Group UK

Erscheinungsjahr

2022

Quelle

MEDLINE

Beschreibungen/Notizen

• 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. WbbB is a structurally unusual retaining glycosyltransferase. Here, the authors show that WbbB forms an Asp232-Kdo adduct prior to transfer to the saccharide acceptor. Therefore, unlike any previously studied glycosyltransferase, WbbB uses the double-displacement mechanism first proposed in 1953.