Kinetic Analysis of the Bisubstrate Cysteine Desulfurase SufS from Bacillus subtilis

Abstract

Cysteine is the major sulfur donor for thio cofactors in bacterial and eukaryotic systems. The first step in sulfur mobilization involves a PLP-dependent enzymatic mechanism. During catalysis, free cysteine is converted into alanine with the concomitant formation of a persulfide bond with the catalytic cysteine residue, thus forming a covalent enzyme intermediate. Cysteine desulfurases in their persulfurated forms serve as donors at the intersection of various cellular sulfur-requiring pathways. Most Gram-positive bacteria, including Bacillus subtilis, contain a cysteine desulfurase gene sufS located adjacent to the gene encoding the proposed Fe-S cluster scaffold SufU. In this work, we identified the participation of SufU as a substrate in the SufS catalytic mechanism. Development of a sensitive method for detection of alanine formed in the SufS reaction enabled the identification of its associated mechanistic features. Steady-state kinetic analysis of alanine formation provided evidence of a double-displacement mechanism (ping-pong) of the cysteine:SufU sulfurtransferase reaction catalyzed by SufS. Results from site-directed mutagenesis of the catalytic cysteine (SufS(C361A)) and iodoacetamide alkylation of SufU support the occurrence of persulfide sulfur transfer steps in the mechanism of SufS.

Functional Analysis of Bacillus subtilis Genes Involved in the Biosynthesis of 4-Thiouridine in tRNA

“…4, left). The affinity of the enzyme for cysteine (K mCys ϭ 29.7 Ϯ 8.7 M), was similar to the values determined for other cysteine desulfurases (15,23,30). The pH dependence activity profile (Fig.…”

“…These results provide further support for involvement of the NifZ active-site cysteine in forming a persulfide intermediate during NifZ's catalytic cycle and the occurrence of a persulfide sulfur transfer step from NifZ to ThiI during the biosynthesis of s 4 U8. Recently, we have described the kinetic mechanism of the bisubstrate cysteine-SufU sulfurtransferase reaction catalyzed by the B. subtilis SufS enzyme (23). The presence of the sulfur acceptor SufU increases the catalytic rate of the SufS reaction by Ͼ100-fold.…”

Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

“…The affinity of the enzyme for cysteine (K m ϭ 4.3 M) is an order of magnitude greater than that of any other cysteine desulfurase found in B. subtilis (see Fig. S4 in the supplemental material) (17,20). As reported for other cysteine desulfurases, the active-site cysteine (Cys325) is essential for YrvO activity (see Fig.…”

“…In previous work, we and others showed that sufS is adjacent to its sulfur acceptor gene, sufU (17,18), coding for a zinc-dependent sulfur transfer protein (19). Both proteins are involved in the formation of Fe-S clusters.…”

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils

“…SufC is an ATPase that has homology with membrane-associated ATPases, SufD participates in Fe acquisition, and SufB is thought to be the site of Fe-S cluster synthesis (12)(13)(14). SufS is a cysteine desulfurase that catalyzes the removal of elemental sulfur from cysteine, producing alanine and a SufS-bound persulfide (15). The persulfide is transferred to SufU, which is a sulfur transfer protein that provides the sulfur to SufBCD (16).…”