1. Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.
- Author
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Bell A, Severi E, Lee M, Monaco S, Latousakis D, Angulo J, Thomas GH, Naismith JH, and Juge N
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Clostridiales genetics, Escherichia coli enzymology, Escherichia coli genetics, Genetic Complementation Test, Humans, Mucins chemistry, Mucins metabolism, N-Acetylneuraminic Acid genetics, N-Acetylneuraminic Acid metabolism, Oxidoreductases genetics, Oxidoreductases metabolism, Bacterial Proteins chemistry, Clostridiales enzymology, N-Acetylneuraminic Acid chemistry, Oxidoreductases chemistry
- Abstract
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 ( Rg NanOx) 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 Rg NanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the Rg NanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of Rg NanOx 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 ., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Bell et al.)- Published
- 2020
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