Abstract
Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).
Highlights
Primarily as a by-product of carbohydrate and lipid metabolism [3, 4]
Overexpression, Purification, and Characterization of GLX2-5—Sequence comparisons of different GLX2 isozymes in Arabidopsis showed that GLX2-5 has a relatively long N-terminal extension that is predicted to target it for localization in the mitochondrion [23]
To overexpress GLX2-5 in E. coli, this N-terminal leader was removed during subcloning to generate an N terminus of the protein, MQIELVP, which is similar to that in cytosolic GLX2-2 [23]
Summary
Primarily as a by-product of carbohydrate and lipid metabolism [3, 4]. MG can react with DNA to form modified guanylate residues [5] and interstrand cross-links [6]. Increased levels of GLX1 and GLX2 RNA and protein have been detected in tumor cells, including breast carcinoma cells [8]. Increased MG levels have been implicated with several complications associated with diabetes mellitus [1, 14, 15], and changes in glyoxalase enzymes have been linked with neurodegenerative disease [16]. Insight into the kinetic mechanism of GLX1 has allowed the development of new classes of mechanism-based, competitive inhibitors that can inhibit tumor growth in vitro and in vivo [12, 19, 20]. S-D-Lactoylglutathione (SLG) appears to be the preferred substrate for GLX2 from most sources, including human, yeast, and plants (26 –33). Glyoxalase II isolated from African trypanosomes prefers thioesters of trypanothione as substrates [34]
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