Methylglyoxal is a naturally produced cytotoxic electrophile, associated with diabetes and ageing. The toxicity of methylglyoxal is thought to be due to attack of nucleophilic centres of macromolecules including DNA, RNA and proteins. The glyoxalase enzyme system serves to detoxify methylglyoxal via a two-step pathway dependent on cellular glutathione. The first step is the conversion of the non-enzymatically formed glutathione-methylglyoxal hemithioacetal to the corresponding thioester by Glyoxalase I.  Glyoxalase II then cleaves the thioester to produce non-toxic D-lactate and regenerate glutathione.

We have determined the structure of GloA2 from Psedudomonas aeruginosa in space group P6₅ 22 (a = b = 51.04 Å, c = 327.1 Å) to 1.95 Å. The metalloenzyme possesses two sequentially identical metal binding sites. Active octahedral coordination of Ni²⁺ at one of the two sites supports previous data designating GloA2 as a Ni²⁺ class glyoxalase I enzyme. Two Zn²⁺ are bound at the second active site separated by 3.28 Å, with a bridging water molecule between them. This is a typical catalytic arrangement for glyoxalase II enzymes. Enzymatic assays confirm that GloA2 consumes S-D-lactoylglutathione in a metal dependent manner and LC-MS confirms that glutathione and lactate are products of the reaction. Our results demonstrate that GloA2 is a bi-functional enzyme, able to perform all of the enzymatically-catalyzed reactions of the methylglyoxal detoxification pathway. Most interestingly, it is able to perform both reactions with the same underlying protein architecture, distinguishing between reactions by the species of metal ion bound.