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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 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-2-deoxy-2,3-dehydro-N-acetylneuraminic acid 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 ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry 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 depended 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.

Antibiotic resistance is a world-wide occurring problem which threatens human health. Without development of any new and effective antibiotics, the rapid growth of antibiotic-resistant bacterial infections could put society in a situation resembling the pre-antibiotic era when a simple lung infection could kill a human being. This thesis presents two venues for targeting antibiotic resistance. Pathogenic bacteria present in mucus rich environments are able to utilize host-derived sialic acid either as an alternative food source or by incorporating sialic acid to their surface glycoconjugates as a way to evade the host´s immune system. Hence, molecular mimicry enables bacteria to secure an ecological niche for survival. Transport of scavenged sialic acid into the cytoplasm of bacteria occurs through specific membrane bound sialic acid transporters. The cell wall is an essential protective barrier for bacteria. The membrane bound enzyme MraY catalyzes the synthesis of lipid I, an intermediate step in the biosynthesis of peptidoglycan, the cell wall of bacteria. This thesis presents work aimed to structurally and functionally characterize sialic acid transporters and MraY for future development of antibacterial drugs. Starting with a broad approach for expression and purification of sialic acid transporters resulted in low-resolution diffracting crystals of the Pasteurella multocida sialic acid TRAP transporter. In addition the X-ray crystallography structure of the sialic acid transporter SiaT from Proteus mirabilis was determined at 1.95 Å resolution in a substrate-bound outward-open conformation revealing a new sodium site. Furthermore, SiaT transporters have been characterized in vivo and the sialic acid specificity has been characterized for SiaT from Staphylococcus aureus. Structural comparison between MraY and the human homologue GPT have highlighted regions where to modify the natural product inhibitor tunicamycin to selectively target MraY. Further characterization of tunicamycin analogues identified potent inhibitors with reduced eukaryotic toxicity.