Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria

Andrew Bell, Emmanuele Severi, Micah O. Lee, Serena Monaco, Dimitrios Latousakis, Jesus Angulo, Gavin H. Thomas, James H. Naismith, Nathalie Juge

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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 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-acetyl-neuraminic acid intermediate and NAD 1 regeneration. The crystal structure of RgNanOx in complex with the NAD 1 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.

Original languageEnglish
Pages (from-to)13724-13736
Number of pages13
JournalThe Journal of Biological Chemistry
Volume295
Issue number40
Early online date15 Jul 2020
DOIs
Publication statusPublished - 2 Oct 2020

Keywords

  • 2,7-anhydro-Neu5AC
  • 2,7-anhydro-Neu5Ac
  • Escherichia coli
  • Escherichia coli (E. coli)
  • Ruminococcus gnavus
  • STD NMR
  • gut microbiota
  • gut symbiosis
  • microbiology
  • mucin glycosylation
  • nuclear magnetic resonance (NMR)
  • oxidation-reduction (redox)
  • oxidoreductase
  • sialic acid
  • sialic acid transporters
  • symbiosis

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