P158 in DJ-1 is essential for cPGA hydrolase activity. (A) The positional relationship of cPGA with N76, H126, and P158 in the DJ-1–cPGA molecular model. (B) The cPGA hydrolytic activity of the N76W and H126A mutants was monitored by a decrease in Abs₂₃₅ (all n = 3). (C) Sequence alignment between human DJ-1 and E. coli YajL. DJ-1 N76 and H126 correspond to I76 and F127 in YajL (shown in red font). Identical and conserved amino acids are shown in black and blue boxes, respectively. (D) The cPGA hydrolytic activity of WT, P158A, or P158∆ DJ-1 was monitored by a decrease in Abs₂₃₅ (all n = 3). (E and F) The folding stability of WT (blue), P158A (light blue), or P158∆ (orange) DJ-1 was measured using a protein thermal shift assay. Representative relative fluorescence unit (RFU) (E) and positive derivative [d(RFU)/dT] curves with melting temperatures (Tm) (F) are shown. The black line indicates the buffer control. (G) The cPGA hydrolytic activity of WT, P158A, and P158∆ YajL was monitored by a decrease in Abs₂₃₅ (all n = 3). Data in B, D, and G are the mean ± SD of three experiments. *P < 0.05 using one-way ANOVA with Dunnett’s multiple comparisons test (B, D, and G).