cPGA hydrolase activity is present in YajL but not the other prokaryotic DJ-1 homologs. (A) Reaction mechanism for DJ-1 hydrolase conversion of cPGA to 3PG. Since Abs235 indicates thioester formation following cPGA reaction with N-acetyl-L-cysteine (NAC), cPGA consumption by DJ-1 can be monitored by a reduction in Abs235. (B) cPGA hydrolase activity was not observed in YhbO, ElbB, or HchA but was present in YajL. cPGA was incubated with DJ-1, YajL, YhbO, ElbB, or HchA for 3 min, followed by reaction with NAC. A reduction in Abs235 is indicative of cPGA hydrolase activity (n = 3). (C) Absorbance spectra of reaction mixtures containing cPGA and WT DJ-1 (blue), YajL (pink), YhbO (light blue), ElbB (orange), or HchA (green) followed by incubation with NAC. The black line corresponds to buffer alone. (D) Phenylglyoxalase activities of YhbO (light blue) and HchA (orange). The PGO-derived Abs250 decreased following incubation with YhbO or HchA. The black line corresponds to buffer alone. (E) The ElbB thermal shift melt curve confirmed that the enzyme was correctly folded. The black line corresponds to buffer alone. (F and G) A decrease in Abs235 is indicative of cPGA consumption following incubation for 3 min with WT or the C106S mutant of DJ-1 (F) or YajL (G) (all n = 3). Data shown in C–E are representative of three individual experiments. Data in B, F, and G are the mean ± SD of three experiments. *P < 0.05 using one-way ANOVA with Dunnett’s multiple comparisons test (B, F, and G). RFU, relative fluorescence unit.
