Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria

Bell, Andrew, Severi, Emmanuele, Lee, Micah O, Monaco, Serena, Latousakis, Dimitrios, Angulo, Jesus, Thomas, Gavin H, Naismith, James and Juge, Nathalie (2020) Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria. The Journal of Biological Chemistry. ISSN 0021-9258

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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 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.

Item Type: Article
Additional Information: Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Faculty \ School: Faculty of Science > School of Biological Sciences
Faculty of Science > School of Pharmacy
Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: LivePure Connector
Date Deposited: 13 Aug 2020 23:58
Last Modified: 18 Sep 2020 23:53
URI: https://ueaeprints.uea.ac.uk/id/eprint/76432
DOI: 10.1074/jbc.RA120.014454

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