G74–G75 comprise an oxyanion hole that supports formation of the substrate–enzyme intermediate connected via C106. (A) Structural model depicting hydrogen bond formation of G74–G75 and A107 with the cPGA carbonyl group. (B) Absorbance spectra when cPGA was incubated with WT (blue), C106S (light blue), A107H (pink), A107I (orange), or A107P (green) DJ-1. Initial reactions were incubated for 3 min, and then N-acetyl-L-cysteine (NAC) was added. The black line indicates the buffer control. (C) The cPGA hydrolytic activity of the indicated DJ-1 mutants was monitored by a decrease in Abs₂₃₅ (all n = 3). (D) Absorbance spectra showing consumption of cPGA by WT (blue), G74S (light blue), or G75S (orange) DJ-1 mutants. (E) The cPGA hydrolytic activity of the indicated DJ-1 mutants was monitored by a decrease in Abs₂₃₅ (all n = 3). (F and G) Thermal shift denaturation curves of WT (blue), G74S (light blue), G75S (orange), C106S (green), or A107P (red) 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 and I) cPGA consumption by WT YajL or the indicated YajL mutants was monitored as in C and E (all n = 3). Data shown in B, D, F, and G are representative of three individual experiments. The mean ± SD of three experiments is shown in C, E, H, and I. *P < 0.05 using one-way ANOVA with Dunnett’s multiple comparisons test (C, E, H, and I).