The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

The human gut symbiont Ruminococcus gnavus scavenges host‐derived N‐acetylneuraminic acid (Neu5Ac) from mucins, by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D nuclear magnetic resonance (NMR), we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-DANA intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram negative and Gram positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionisation spray mass spectrometry (ESI-MS) that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolise 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry (DSF) analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli was dependent on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP‐STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D2O/H2O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP‐STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket.