The nickel-pincer nucleotide (NPN) is an organometallic cofactor that features Ni(II) tri-coordinated to a pyridinium mononucleotide in a planer configuration using two thioacid sulfur atoms and a covalent bond to a ring carbon atom. This arrangement, where a metal ion is held in a rigid planar conformation by two ligands attached to an aromatized ring and by an atom within the ring, is termed a pincer complex. Pincer complexes are well known in synthetic organic chemistry; however, NPN is one of only two known examples of such a complex in biology. NPN was first reported in 2015 in Lactobacillus plantarum lactate racemase (LarA), an enzyme which interconverts the two stereoisomers of lactate. This organism requires three gene products (LarB, LarE, and LarC) to synthesize the cofactor from nicotinic acid adenine dinucleotide (NaAD), typically considered a precursor to NAD.Computational analyses had generated competing proposals for the reaction mechanism of lactate racemization. Through biochemical analysis of LarA, I demonstrated that it operates by a proton-coupled hydride transfer mechanism during which lactate is transiently oxidized to pyruvate as a hydride transfers onto NPN. Hydride return from NPN can occur to either face of the planar and pro-chiral pyruvate, restoring lactate while achieving racemization.The first synthetic step in NPN synthesis is catalyzed by LarB, an NaAD carboxylase/hydrolase. LarB carboxylates C5 of the nicotinic acid moiety of NaAD and hydrolyzes the phosphoanhydride, releasing AMP and the product, pyridinium-3,5-biscarboxylic acid mononucleotide. I was able to crystallize and structurally characterize LarB and its complexes with NAD+ and AMP. My structural and biochemical experiments demonstrate that LarB utilizes CO2 for carboxylation and my work suggests that the enzyme activates the substrate by forming a covalent bond between a cysteine residue and C4 of the nicotinic acid. The resulting dihydropyridine then undergoes carboxylation by nucleophilic addition to CO2 followed by deprotonation and rearomatization with expulsion of the cysteine.The second step in the synthesis pathway utilizes LarE, a sulfur insertase that uses ATP to activate the substrate by adenylylation then sacrificially donates a sulfur atom originating from a cysteine residue, leaving behind a dehydroalanine (Dha) residue in the protein. Two cycles of this process are needed to create pyridinium-3,5-bisthiocarboxylic acid, which is then metalated by LarC to form NPN. The requirement for two equivalents of the 31 kDa LarE protein for the synthesis of a single molecule of NPN represents an enormous energy investment by the cell. My work has suggested a route for regeneration of the cysteine residue from Dha. I have shown that CoA persulfide robustly reacts with Dha in the inactivated LarE, forming the LarE Cys-CoA form of the enzyme, and subsequent reduction restores the native form of LarE. Notably, LarE has a high affinity for CoA and the LarE Cys-CoA disulfide is known to be an active form of the enzyme in vitro when exogenous CoA is provided at physiologically relevant concentrations. Nevertheless, the in vivo relevance of this reaction is yet to be elucidated.
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