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

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. 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 beta-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-beta-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.

Automated summary

WbbB, a structurally unusual retaining glycosyltransferase, forms an Asp232-Kdo adduct prior to transfer. This double-displacement mechanism, first proposed in 1953, differs from other glycosyltransferases. Structural analysis shows that WbbB repositions the Kdo into a second sub-site for transfer. The study suggests that retaining glycosyltransferases can use double-displacement mechanisms.