Pseudomonas aeruginosa contains multiple glyoxalase I-encoding genes from both metal activation classes Nicole Sukdeo, John F. Honek ⁎ Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Received 25 January 2007; received in revised form 3 April 2007; accepted 11 April 2007 Available online 20 April 2007 This paper is dedicated to the memory of Professor Donald Creighton Abstract 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 Zn 2+ -dependent or non-Zn 2+ dependent (Ni 2+ -/Co 2+ -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-Zn 2+ dependent glyoxalase I enzymes and the gloA3 gene remarkably encodes a Zn 2+ -dependent homolog. To our knowledge this is the first report of a eubacterial species with several GlxI encoding genes, and also of an organism possessing GlxI enzymes from both metal activation classes. © 2007 Elsevier B.V. All rights reserved. Keywords: Glyoxalase I; Pseudomonas aeruginosa; Homologues; Metal activation; Nickel; Zinc 1. Introduction The glyoxalase (Glx) enzyme system consists of two metalloenzymes which detoxify cytotoxic methylglyoxal (MG) to its corresponding α-hydroxyacid. GlxI (S-D-lactoyl- glutathione methylglyoxal lyase (isomerizing), EC 4.4.1.5) initially accepts MG as a non-enzymatically formed hemi- thioacetal of glutathione (GSH) or in some cases trypa- nothione ( N 1 , N 8 -bis(glutathionyl)spermidine, abbreviated TSH) [1–3]. GlxI catalyzes a proton transfer, which converts incoming hemithioacetal to the corresponding thioester (Fig. 1). The metallohydrolase GlxII (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) regenerates the thiol co-substrate and the α-hydroxyacid, which in the case of MG detoxification, is D-lactate. This detoxification pathway is a contemporary focus of investigation across diverse biological contexts [4–9]. For example, MG toxicity is recognized as a participant in a broad spectrum of human chronic disease states [10–13]. Regulatory roles of MG have recently been discovered, conferring novel functions upon this metabolite. Studies of the glyoxalase system in Escherichia coli implicated the GlxI product S-D- lactoylglutathione as a physiologically significant activator of potassium efflux systems (KefB and KefC) that confer protection against the toxic effects of electrophiles (i.e. MG) [14–16]. MG appears to function as a transcriptional activator and in kinase cascade activation, implying a role for the Glx system in regulatory/adaptive intracellular circuits [5,6,17]. The prospect of the Glx system being assigned additional roles besides detoxification may provide additional clarification for the nearly ubiquitous biological distribution of these enzymes. Glx system components also appear to be involved in cellular processes specific to microbial species. Interestingly a putative www.elsevier.com/locate/bbapap Abbreviations: Glx, Glyoxalase; GlxI, Glyoxalase I; GlxII, Glyoxalase II; MG, methylglyoxal; GSH, glutathione; TSH, N 1 ,N 8 -bis(glutathionyl)spermi- dine; PCR, polymerase chain reaction; TB, terrific broth; DPA, 2,6- pyridinedicarboxylic acid; IPTG, isopropyl β-D-thiogalactoside; DTT, dithio- threitol; KPB, potassium phosphate buffer; PMSF, phenylmethylsulphonyl fluoride; SDS/PAGE, sodium dodecylsulphate polyacrylamide gel electropho- resis; ESMS, electrospray ionization mass spectrometry; ICP-MS, inductively coupled plasma mass spectrometry; PAR, 4-(2-pyridylazo)resorcinol; EDTA, ethylenediaminetetraacetic acid ⁎ Corresponding author. E-mail address: jhonek@uwaterloo.ca (J.F. Honek). Biochimica et Biophysica Acta 1774 (2007) 756 – 763 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.04.005