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Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria

DOI: 10.1074/jbc.RA120.014454 DOI Help

Authors: Andrew Bell (Quadram Institute Bioscience) , Emmanuele Severi (University of York) , Micah O. Lee (University of Oxford) , Serena Monaco (University East Anglia) , Dimitrios Latousakis (Quadram Institute Bioscience) , Jesus Angulo (University of East Anglia) , Gavin H. Thomas (University of East Anglia) , James Naismith (University of Oxford) , Nathalie Juge (Quadram Institute Bioscience)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: July 2020

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.

Journal Keywords: 2,7-anhydro-Neu5AC; oxidoreductase; Ruminococcus gnavus; mucin glycosyation; gut symbiosis; microbiology; oxidation-reduction (redox); Escherichia coli (E. coli); nuclear magnetic resonance (NMR); sialic acid

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography