Abstract
ABSTRACT
Posttranslational sulfur incorporation into peptide backbones is increasingly reported. These efforts broaden our understanding of thioamides, which have distinct conformational and hydrogen‐bonding properties compared with amides. YcaO‐catalyzed thioamidation of the ribosomal protein uL16 reportedly occurs across thousands of Pseudomonadota. Here, we identify the cysteine desulfurase IscS, a crucial component of iron–sulfur cluster assembly, as the direct sulfur donor for uL16 thioamidation. Genetic deletion, in vitro reconstitution, kinetic analysis, sulfide scavenging, and mutational studies support this conclusion. Our data support a mechanism in which the IscS persulfide attacks the
O
‐phosphorylated uL16 backbone amide to form an IscS–uL16 disulfide intermediate. This labile disulfide is rapidly reduced, and the resulting hemiorthothioamide collapses with phosphate release to give the thioamide. IscS binds uL16 with mid‐nanomolar affinity and can be displaced by other IscS‐dependent sulfur acceptors. IscS promotes formation of the 2:2 YcaO–uL16 complex from the otherwise predominant 1:1 complex. Structural and mutational studies identify uL16 residues important for catalytically competent enzyme–substrate interactions. These findings define the sulfur delivery pathway for uL16 thioamidation and reveal an additional role of the IscS persulfide beyond iron–sulfur cluster assembly, suggesting it serves as another nucleophile for YcaO‐catalyzed backbone modification.