Enzymes, constructed only of the 20 canonical amino acids, catalyse a remarkable range of reactions. Nevertheless, in order to expand their scope of catalytic properties, enzymes take advantage of metal ions, organic metabolites, or metal organic complexes, known as cofactors.

One such metal organic complex, the nickel-pincer nucleotide (NPN), has been discovered by Benoit Desguin a few years ago in lactate racemase (LarA), an enzyme interconverting both lactate enantiomers. This cofactor combine the properties of a scaffold derived from nicotinic acid, with a nickel ion coordinated by two sulfurs and forming a nickel-carbon bond with the C4 position of the pyridinium ring. It has been shown that its function is the reversible capture of a hydride, similarly to nicotinamide adenine dinucleotide (NAD), Nevertheless, the presence of carbon-nickel bond endows NPN with unique properties. Quoting Prof. Deborah Zamble : “…metal carbon bonds are rare in biology, and this is the first one found to include a nicotinic-based cofactor…why an organism would go to the trouble of making such a fancy cofactor?. This is the central question of the research group.

We discovered that LarA is not the only enzyme using the NPN cofactor, but that LarA homologs (LarAHs) form a very diverse enzymatic superfamily found in one out of eight bacterial or archaeal species, some members being even present in eukaryotes, i.e. oomycetes, diatoms, and microalgae. A first in vitro analysis of some of these homologs showed than they catalyse racemization or epimerization of a variety of substrates: short aliphatic α-hydroxyacids, malate, hydroxyglutarate, phenyllactate, gluconate and many others. We have some indications that some of these homologs could be involved in other types of yet unknown epimerization reactions.

Shortly after the discovery of NPN, we showed that NPN biosynthesis involves LarB, LarC and LarE and nicotinic acid adenine dinucleotide (NaAD) as a precursor. LarB starts by catalysing the carboxylation of the nicotinic ring that is accompanied by hydrolysis of the phosphoanhydride bond. LarE converts both the carboxylate groups into thiocarboxylate groups by an ATP-dependent sacrificial sulfur insertion. As the two sulfur atoms inserted are derived from one cysteine of LarE, two LarE proteins are required for the synthesis of one NPN molecule. Finally, nickel-containing LarC catalyses the CTP-dependent nickel metalation between one sulfur atom and the carbon, generating NPN. LarC behaves as a single turnover enzyme in vitro, similarly to LarE. Finally, in case of covalent NPNylation, NPN reacts spontaneously with a lysine of LarA, forming a thioamide bond. Many of these biosynthetic steps are still mysterious, and some of our research projects aim to elucidate the reactions of this pathway.

Furthermore, the genes coding for the NPN-biosynthesis enzymes were also identified without a larA gene in another 15% of the studied bacterial and archaeal genomes, suggesting that enzymes that are not homologous to LarA use NPN as well. These enzymes could catalyse other unknown reactions requiring NPN, yet not necessarily involving epimerization or racemization reactions.

In summary, our group aim to decipher the biosynthetic pathway towards NPN and discover the scope of utilization of this cofactor in nature.