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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.