The catalytically critical E18 is stringently regulated by R28. (A) Distance plot of residues that form the cPGA-binding site in DJ-1 based on the unstrained 50-ns MD simulation. (B) Absorbance spectra of reaction mixtures containing cPGA and WT (blue), E18A (light blue), E18Q (pink), or R28A (orange) DJ-1. Initial reactions were incubated for 3 min, and then N-acetyl-L-cysteine (NAC) was added. The thioester peak at Abs₂₃ decreases if the DJ-1 mutants can hydrolyze cPGA. The black line corresponds to buffer alone. (C) The cPGA hydrolytic activity of WT, E18A, E18Q, or R28A DJ-1 was monitored by a decrease in Abs₂₃₅ (all n = 3). (D) Absorbance spectra are as in B following incubation with WT (blue), E15A (light blue), or R48A (orange) DJ-1. (E) The cPGA hydrolytic activity of WT, E15A, or R48A DJ-1 was monitored by a decrease in Abs₂₃₅ (all n = 3). (F and G) Thermal shift denaturation curves of WT (blue), E15A (light blue), E18A (orange), E18Q (red), or R28A (green) DJ-1. Representative relative fluorescence unit (RFU) (F) and positive derivative [d(RFU)/dT] curves with melting temperatures (Tm) (G) are shown. The black line indicates the buffer control. (H) Distance plot of residues that form the cPGA-binding site in YajL based on the unstrained 50-ns MD simulation. (I) The cPGA hydrolytic activity of WT or the indicated YajL mutants was monitored by a decrease in Abs₂₃₅ (all n = 3). Data shown in B, D, F, and G are representative of three individual experiments. Data in C, E, and I are the mean ± SD of three experiments. *P < 0.05 using one-way ANOVA with Dunnett’s multiple comparisons test (C, E, and I).