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

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.

Mechanism and linkage specificities of the dual retaining β-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis

“…2 C ). A similar tendency to form Kdo-adducts during protein expression was observed with WbbB (GT99) nucleophile variants ( 19 ). Incubation of the D160N variant with CMP-Kdo resulted in an apparent increase in the relative abundance of species containing the covalent Kdo adduct ( Fig.…”

“…Previous structural and functional analyses of β-Kdo GTs led to a prediction that an absolutely conserved aspartate residue (from the invariant QXXXD motif in the active site) acts as the catalytic nucleophile for these enzymes; in KpsC-N Ec , this residue is Asp160 ( 5 , 9 ). The involvement of the corresponding aspartate residue in the retaining WbbB β-Kdo GT was verified experimentally ( 19 ). To further investigate the role of this residue in KpsC, WT KpsC-N Ec and variants with Asp160 substitutions were examined for their ability to form covalent adducts with Kdo.…”

“…Comparison of the Kdo adduct from KpsC-N Ec D160N with the equivalent WbbB GT99 D232N (PDB ID 8CSC ) ( 19 ) reveals very similar positioning and orientation of the Kdo adduct in each active site, despite none of the coordinating groups being conserved between the two enzymes ( Figs. 4 F and S3 B ).…”

“…An alternative double-displacement mechanism, analogous to the well-established mechanism of retaining glycoside hydrolases (reviewed in ( 17 )), has been proposed for one GT6-family enzyme ( 18 ), but experimental support for broader involvement of this mechanism in retaining GTs was limited. We recently provided experimental evidence that another β-Kdo GT (WbbB, a GT99 prototype from lipopolysaccharide O-antigen biosynthesis in Raoultella and Klebsiella ) uses a double-displacement mechanism ( 19 ). KpsC is a distant homolog of WbbB and conserves three critical catalytic residues found in WbbB; an aspartate that serves as the nucleophile and forms an Asp–Kdo adduct, a histidine that protonates the CMP phosphate leaving group, and a glutamate that acts as a base to activate the incoming acceptor.…”

In silicomodelling of the function of disease-related CAZymes

“…In a second step, an acceptor molecule attacks the anomeric carbon, breaking the glycosyl-enzyme covalent bond and forming a new glycosidic bond, with overall retention of stereochemistry. A similar mechanism has been recently proved by structural and mass spectrometry experiments on a GT99 [ 34 ]. There is theoretical evidence of a double-displacement mechanism for GT6 enzymes [ 35 , 36 ], although experimental detection of the covalent intermediate remains challenging [ 37 , 38 ].…”