LAUSR

We recently reported the production of a large quantity of reactive persulfides including cysteine hydropersulfide (CysSSH) in both prokaryotes and eukaryotes. Also, these reactive persulfides reportedly show critical functions for regulation of redox signaling. Currently, we found that Escherichia coli (E.coli) and mammalian cysteinyl-tRNA synthetases (CARSs) effectively produce CysSSH from the substrate L-cysteine. However, the characterization of the CARSs has not been fully elucidated. Our present study now showed that the CysSSH synthase activity of E. coli CARS (EcCARS) depends on pyridoxal phosphate (PLP) but not on ATP and tRNA which required for the activity of cysteinyl-tRNA synthetase. LC-MS analysis and Mascot data search revealed that PLP-binding sites on EcCARS are lysine (K) residues including 73KIIK76 and 266KMSK269 motifs. Intriguingly, the KIIK and KMSK motifs are highly conserved among E.coli and mammalian species. The EcCARS mutants with a single mutation (K73A, K76A, K266A, and K269A) and double mutations (K73/76A and K266/269A) at theses KIIK/KMSK motifs showed significantly decreased CysSSH synthase activity. Intriguingly, these mutants possessed the intact protein synthesis, as assessed by the cell-free protein synthesis assay. Furthermore, we constructed mutant (C28D) of cysteine (Cys28) that is a Zn2+ ligand essential for the activity of cysteinyl-tRNA synthetase. Although the C28D mutant was deficient in the activity of protein synthesis, its CysSSH synthesis activity remained intact. Taken together, we clarified for the first time that EcCARS has a unique moonlighting function to co-translationally produce CysSSH independent of its catalytic function for the aminoacyl-tRNA biosynthesis. The elucidation of the mechanism of CysSSH synthesis by CARSs may contribute to the understanding of redox signalling mediated by CysSSH.