Chemo-enzymatic synthesis of NPN cofactor taking advantage of ADP-ribosyl cyclase and LarC cyclometallase promiscuous activities.

The nickel-pincer nucleotide cofactor (NPN) is a widespread organometallic cofactor required for lactate racemase (LarA) and for α-hydroxy acid racemases and epimerases of the LarA superfamily. Its biosynthesis, which starts with nicotinic acid adenine dinucleotide (NaAD), requires three enzymes: LarB, LarC, and LarE, and can be performed in vitro with purified enzymes. Nevertheless, as LarE and LarC are single turnover enzymes, the in vitro NPN biosynthesis requires huge amounts of enzymes (particularly 2 equivalents of LarE), which hampers the study of NPN and of NPN-dependent enzymes. By using adenosine diphosphate (ADP)-ribosyl cyclase (ARC), we exchanged the nicotinamide moiety in NAD⁺ with synthetic pyridine-3,5-bisthiocarboxylic acid in order to synthesize the novel intermediate pyridinium-3,5-bisthiocarboxylic acid adenine dinucleotide (P2TAD). The latter could be produced at a multimilligram scale allowing its characterization by Nuclear Magnetic Resonance (NMR) and mass spectrometry. Interestingly, P2TAD could directly be used by LarC in order to generate the NPN cofactor, bypassing both LarB and LarE. Globally, a new chemoenzymatic route towards NPN was developed via the intermediate P2TAD, which should facilitate the biochemical and biotechnological investigations on NPN binding enzymes.