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The Structurally Unusual Retaining β‐Kdo Glycosyltransferase WbbB Uses a Double‐Displacement Mechanism with an Intermediate Adduct Rearrangement Step

WbbB is a modular, trifunctional glycosyltransferase (GT) which synthesizes lipopolysaccharide O‐antigen in Raoultella terrigena. The two C‐terminal WbbB GT domains build the polysaccharide repeat, while the N‐terminal GT domain acts as a terminator, adding a β‐Kdo (3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid) residue to O3 to rhamnose as required for O‐antigen export. This N‐terminal GT domain is highly unusual in sequence and was not recognized as a GT by bioinformatics tools. The structure shows the dual Rossmann‐fold motifs characteristic of GT‐Bs, but with extensive deletions, insertions and rearrangements result in a unique architecture. The CMP‐binding site, however, retains motifs homologous to GT‐B sialyltransferases. WbbB is a retaining GT, transferring Kdo from the CMP‐®‐Kdo donor with net retention of anomeric configuration. All well‐characterized retaining GTs seem to employ a front‐side SNi substitution mechanism. This is somewhat surprising, as analogous retaining glycosyl hydrolases use a double‐displacement mechanism; here an essential catalytic acid residue attacks the donor saccharide to form a covalently bonded intermediate, with a second acidic residue acting as a general base to hydrolyse this adduct. We show, using mass spectrometry, that WbbB forms a Kdo adduct with Asp232 and its variants (D232N or D232C), while a D232A variant is both wholly inactive and forms no adduct. The x‐ray structure of D232N CMP‐®‐Kdo donor complex shows that the anomeric carbon of the donor is inaccessible to any potential acceptor but is instead positioned immediately in contact with, and in line with, the carboxylate of Asp232. Structures of D232N‐ and D232C‐Kdo adducts show that the Kdo adduct is rearranged into a second half‐site, interacting with a distinct set of catalytic residues. A ternary complex of D232C‐Kdo plus a synthetic acceptor shows that Glu158 forms direct hydrogen bonds with O3 of rhamnose, positioned adjacent to and in‐line with the anomeric carbon of Kdo, activating it for attack. Glu158 is also essential, with variants being wholly inactive. Together, this shows that WbbB uses a glycosyl hydrolase‐like double‐displacement mechanism. We propose that crowding around the anomeric carbon by the carboxylate group likely precludes retaining ulosonic acid transferases using the more straightforward SNi mechanism, while adduct rearrangement avoids having the acceptor compete with the leaving nucleotide for access to the anomeric carbon of the donor.