Proposed reaction mechanism for DJ-1–mediated cPGA hydrolysis. (A) The reaction is initiated following the formation of hydrogen bonds between the substrate (cPGA) and E18, G74–75, and A107 in the DJ-1 active site (step 1). Attack of C106 on the carbonyl carbon yields the first tetrahedral intermediate that is stabilized by the oxyanion hole comprised by G74 and G75 (step 2). Reformation of the carbon-oxygen double bond leads to formation of the acyl enzyme intermediate via a thioester linkage (step 3). Water then attacks the carbonyl carbon of the acyl enzyme (step 4) to form the second tetrahedral intermediate, which is similarly stabilized by the oxyanion hole as in step 2 (step 5). Lastly, reformation of the carbon-oxygen double bond restores the free enzyme and releases the 3PG product (step 6). (B) Schematic diagram showing how E18 interacts with cPGA. Hydrogen bond formation between E18 and the cPGA hydroxyl group is assisted by the intermolecular salt bridge formed with R28.