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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-acetylneuraminic acid 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 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.

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives., Graphical abstract Membrane-enclosed multienzyme (MEME) approach for the production of 2,7-anhydro-sialic acid derivatives.Image 1, Highlights • A novel membrane enclosed multiple enzyme approach was developed for the production of 2,7-anhydro-sialic acid derivatives. • 2,7-anhydro-Neu5Ac was produced in high-yield and high-purity. • Neu5Gc-rich glycoprotein was produced using a one-pot two-step enzymatic synthesis. • The method allowed the synthesis of a novel compound, 2,7-anhydro-Neu5Gc.