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 language | English |
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Pages (from-to) | 13724-13736 |
Number of pages | 13 |
Journal | The Journal of Biological Chemistry |
Volume | 295 |
Issue number | 40 |
Early online date | 15 Jul 2020 |
DOIs | |
Publication status | Published - 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
Profiles
-
Jesus Angulo
- School of Chemistry, Pharmacy and Pharmacology - Honorary Senior Lecturer
- Pharmaceutical Materials and Soft Matter - Member
Person: Honorary, Research Group Member