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The NadABC pathway is involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is a dominant pathway in bacteria. The conversion of l-aspartate to quinolinic acid is initiated by the l-aspartate oxidase NadB, which catalyzes the formation of iminoaspartate that is used by quinolinate synthase NadA in a condensation reaction with dihydroxyacetone phosphate to produce quinolinic acid. NadA is a [4Fesingle bond4S] cluster-containing enzyme that is indispensable in the production of NAD. In B. subtilis, the cysteine sulfurtransferase nifS gene is located in genomic proximity to the nad genes, and its expression is regulated by NadR based on the availability of nicotinic acid. Inactivation of nifS leads to inactivation of the NAD pathway and, consequently, nicotinic acid auxotrophy. In this study, we explored the hypothesis that NifS’ involvement in NAD biosynthesis is associated with its role in the maturation of NadA [4Fesingle bond4S] cluster. We showed through in vitro reconstitution experiments that NifS is catalytically competent in promoting cluster assembly onto apo-NadA and that the rate of reactivation depends on the rate of sulfur mobilization. Furthermore, the activity of NifS in sulfur mobilization is modulated by Apo-NadA. Under conditions of cluster synthesis, apo-NadA enhances the turnover rate of NifS. This phenomenon is not observed for YrvO, NifZ, and SufSU, the other three cysteine sulfurtransferases in B. subtilis. This work provides biochemical evidence for the requirement of a dedicated cysteine desulfurase in the maturation of specialized Fesingle bondS enzymes. 

The biological synthesis of iron–sulfur (Fe–S) clusters requires dedicated pathways involved in the recruitment and activation of Fe and S for cluster assembly with subsequent transfer of preformed clusters to acceptor proteins. Several pathways have been described that include various numbers and types of biosynthetic components, although all of them share the same basic principles for [Fe–S] cluster formation and delivery to target proteins. The NifUS system was discovered and first described in studies involving the model diazotroph Azotobacter vinelandii . It has a dedicated role in serving as the starting point for the activation of [Fe–S] cluster-containing proteins specifically involved in biological nitrogen fixation. NifS is a pyridoxal-5′-phosphate containing l -cysteine-dependent sulfur transferase that delivers activated sulfur to the three-domain NifU, which not only serves as a scaffold for the construction of [2Fe–2S] and [4Fe–4S] clusters but also participates in their delivery to various target proteins involved in nitrogen fixation. Interestingly, analysis of sequenced genomes reveals that the three-domain NifU and NifU-like encoded proteins are not limited to diazotrophs, suggesting a broader role for this system in [Fe–S] cluster biogenesis in other organisms. The colocalization of adjacent nifU and nifS encoding sequences in most of these genomes also provides a strong indication for the involvement of the NifU–NifS [Fe–S] cluster assembly and delivery toolkit for activation of [Fe–S] cluster-containing proteins in a variety of organisms that do not fix nitrogen. 

Sulfur-containing biomolecules such as [Fe-S] clusters, thiamin, biotin, molybdenum cofactor, and sulfur-containing tRNA nucleosides are essential for various biochemical reactions. The amino acid l-cysteine serves as the major sulfur source for the biosynthetic pathways of these sulfur-containing cofactors in prokaryotic and eukaryotic systems. The first reaction in the sulfur mobilization involves a class of pyridoxal-5′-phosphate (PLP) dependent enzymes catalyzing a Cys:sulfur acceptor sulfurtransferase reaction. The first half of the catalytic reaction involves a PLP-dependent single bondS bond cleavage, resulting in a persulfide enzyme intermediate. The second half of the reaction involves the subsequent transfer of the thiol group to a specific acceptor molecule, which is responsible for the physiological role of the enzyme. Structural and biochemical analysis of these Cys sulfurtransferase enzymes shows that specific protein-protein interactions with sulfur acceptors modulate their catalytic reactivity and restrict their biochemical functions.