The nickel-pincer nucleotide (NPN) cofactor is an organometallic cofactor critical for the activity of lactate racemase (LarA) and other racemases and epimerases. This dissertation explores the biosynthesis, mechanistic roles, and detection strategies for the NPN cofactor, offering new insights into its biochemical and structural characteristics.The first chapter introduces the pathway for NPN biosynthesis in Lactiplantibacillus plantarum, detailing the roles of LarB, LarC, and LarE enzymes. Subsequent chapters elucidate mechanisms underlying key steps of NPN assembly, including the identification of intermediates in LarB-catalyzed reactions and the exploration of sulfur transfer pathways mediated by LarE. The potential for IscS to enhance LarE activity in Latilactobacillus sakei is also examined, exploring alternative mechanisms for sustaining enzymatic activity. In addressing challenges in studying NPN-dependent enzymes, novel methodologies were developed to enable detection and functional characterization. A circular dichroism spectroscopy assay was optimized for kinetic analysis of LarA and LarA homologs, while sulfonyl azide-based resins were employed to selectively isolate NPN-bound proteins from cell lysates. Additionally, efforts to detect the postulated nickel-hydride intermediate in LarA using 1H-NMR spectroscopy highlight the complexities of probing transient intermediates in catalytic cycles. This work advances our understanding of the biosynthesis, function, and detection of the NPN cofactor, providing a foundation for investigating its role in microbial metabolism and uncovering potential applications in biocatalysis and biotechnology.
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