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The glyoxalase (Glx) system is a critical detoxification enzyme system that is widely distributed in prokaryotic and eukaryotic organisms. Glyoxalase I (GlxI), the first enzyme in the system, is a divalent metal-ion dependent lyase (isomerizing), and its homologs have recently been categorized into two metal activation classes which are either Zn²⁺-dependent or non-Zn²⁺ dependent (Ni²⁺-/Co²⁺-activated). The latter class encompasses enzymes of predominantly bacterial origin. We have identified two genes in Pseudomonas aeruginosa PAO1 encoding glyoxalase I enzymes in addition to the gloA1 sequence recently reported and characterized. The gloA1 and gloA2 genes encode non-Zn²⁺ dependent glyoxalase I enzymes and the gloA3 gene remarkably encodes a Zn²⁺-dependent homolog. To our knowledge this is the first report of a eubacterial species with several GlxI encoding genes, and also of an organism …