Format of Original
American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry
Original Item ID
Glyoxalase 2 is a β-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 1H 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 kcat and Km values of 129 ± 10 s-1 and 391 ± 48 μm, 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. 1H 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-Å 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).
Marasinghe, Gishanthi P. K.; Sander, Ian M.; Bennett, Brian; Periyannan, Gopal R.; Yang, Ke-Wu; Makaroff, Christopher A.; and Crowder, Michael W., "Structural Studies on a Mitochondrial Glyoxalase II" (2005). Physics Faculty Research and Publications. 79.
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