Abstract
Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarETm) and show that it uses these three conserved cysteine residues to bind a [4Fe-4S] cluster that is required for sulfur transfer. Notably, we found LarETm retains all side chain sulfur atoms, in contrast to LarELp. We also demonstrate that when provided with L-cysteine and cysteine desulfurase from Escherichia coli (IscSEc), LarETm functions catalytically with IscSEc transferring sulfane sulfur atoms to LarETm. Native mass spectrometry results are consistent with a model wherein the enzyme coordinates sulfide at the nonligated iron atom of the [4Fe-4S] cluster, forming a [4Fe-5S] species, and transferring the noncore sulfide to the activated substrate. This proposed mechanism is like that of TtuA that catalyzes sulfur transfer during 2-thiouridine synthesis. In conclusion, we found that LarE sulfur insertases associated with NPN biosynthesis function either by sacrificial sulfur transfer from the protein or by transfer of a noncore sulfide bound to a [4Fe-4S] cluster.