Publications

• The putative Escherichia coli dehydrogenase YjhC metabolises two dehydrated forms of N-acetylneuraminate produced by some sialidases.

  Kentache T., Thabault L., Peracchi A., Frederick R., Bommer G.T., Van Schaftingen E.

  Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deo ... >> More

  Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 β-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT. << Less

  Biosci. Rep. 40:0-0(2020) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria.

  Bell A., Severi E., Lee M., Monaco S., Latousakis D., Angulo J., Thomas G.H., Naismith J.H., Juge N.

  The human gut symbiont <i>Ruminococcus gnavus</i> scavenges host-derived <i>N</i>-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into <i>R. gnavus</i> ATCC 29149 before being converted back to Neu5Ac fo ... >> More

  The human gut symbiont <i>Ruminococcus gnavus</i> scavenges host-derived <i>N</i>-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into <i>R. gnavus</i> 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 (<i>Rg</i>NanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-<i>N</i>-acetylneuraminic acid intermediate and NAD⁺ regeneration. The crystal structure of <i>Rg</i>NanOx in complex with the NAD⁺ cofactor showed a protein dimer with a Rossman fold. Guided by the <i>Rg</i>NanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of <i>Rg</i>NanOx 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 <i>Escherichia coli</i> homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that <i>E. coli</i> 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 <i>E. coli</i> mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in <i>E. coli</i> 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 <i>E. coli</i>. << Less

  J. Biol. Chem. 295:13724-13736(2020) [PubMed] [EuropePMC]