Project Summary/Abstract The nickel-pincer nucleotide (NPN) cofactor is a recently identified coenzyme discovered in lactate racemase (LarA) but found more widely in 2-hydroxyacid racemases and epimerases. Synthesis of the cofactor requires the sequential action of three accessory proteins: LarB carboxylates the pyridinium ring at C5 and hydrolyzes the phosphoanhydride of nicotinic acid adenine dinucleotide (NaAD), LarE is an ATP-dependent enzyme that converts the two pyridinium ring carboxylates into thiocarboxylates either by sacrificial loss of a protein cysteinyl sulfur atom or by a mechanism involving a [4Fe-4S] cluster with a non-core sulfide, and LarC inserts nickel (forming C-Ni and S-Ni bonds) in a CTP-dependent reaction. Gene homologs encoding these four proteins are widely distributed in microorganisms associated with the human microbiome and among human pathogens. The long-term objective of the effort described here is to significantly advance our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor. Two specific aims will achieve this objective: (1) the components of the NPN biosynthetic pathways will be structurally and mechanistically characterized and (2) new roles of the NPN cofactor will be characterized beyond its function in lactate racemase. Investigations of LarB will define the structure of an NaAD- and CO2-bound variant by crystallography and confirm the formation of a covalent cysteinyl-pyridinium intermediate by stopped-flow UV- vis spectrophotometry. In vivo analyses will test the cellular ability to reverse the sacrificial loss of cysteinyl sulfur from one type of LarE. The [4Fe-4S] cluster-containing version of LarE will be structurally characterized by crystallography. The hypothesis for a third, persulfide-containing form of LarE will be evaluated. The structure and dynamics of intact LarC will be solved by cryo-electron microscopy. Alternatively, the N-terminal domain of LarC will be defined by crystallography (complementing the known C-terminal structure) to better delineate the enzyme’s single-turnover, CTP-dependent mechanism of nickel insertion. Additional LarA homologs will be structurally and functionally defined. Examples that tightly bind larger substrates will be used to probe open questions regarding the NPN-dependent enzyme reaction mechanism. Other examples are predicted to contain both NPN and an iron-sulfur cluster whose function will be investigated. In addition, non-LarA-like proteins that bind NPN will be identified and characterized. The findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel-pincer nucleotide cofactors in bacteria, including those important to human health, with implications for identifying potential antimicrobial drug targets.