Fig. 3

Computational modeling of EcCARS structure, and CysS–(S) _n –H biosynthesis by CARS1/2. a A molecular docking model of PLP-bound EcCARS generated by SwissDock using the crystal structure of EcCARS (PDB ID: 1LI5). Cysteinyl-tRNA is placed by superimposing the crystal structure of the EcCARS-Cysteinyl-tRNA binary complex (PDB ID: 1U0B) to the docking model. b, c PLP-dependent CysSSH and CysSSSH biosynthesis by mouse CARS1 and human CARS2. CysSSH and CysSSSH production was quantified by means of HPE-IAM labeling LC-MS/MS analysis in the reaction of recombinant mouse CARS1 and human CARS2 (200 μg/ml each) with 25 μM L-cysteine in the presence or absence of 100 μM PLP (37 °C, 2 h). The data are means ± s.d. (n = 3). *P < 0.01. d Concentration-dependent effects of PLP on CysSSH and CysSSSH production by recombinant human CARS2. Human CARS2 (200 μg/ml) reacted with 25 μM cysteine in the presence of 0, 10, 50, or 100 μM PLP at 37 °C for 30–120 min. No appreciable cysteine persulfide production was detected in the reaction mixture of cysteine and PLP alone as long as no >100 μM PLP was used. e Precisely quantitative analysis for PLP bound to human CRAS2. Human CARS2 treated with various concentrations of PLP (d) at 37 °C for 1 h was reacted with DNPH to form PLP-DNPH adduct, followed by quantification by LC-ESI-MS/MS analysis