Structural studies on a mitochondrial glyoxalase II.
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).
Marasinghe
Gishanthi P K
GP
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
Sander
Ian M
IM
Bennett
Brian
B
Periyannan
Gopalraj
G
Yang
Ke-Wu
KW
Makaroff
Christopher A
CA
Crowder
Michael W
MW
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000648:ultrastructure; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D015870:Gene Expression; D006801:Humans; D007501:Iron; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000032:analysis; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase
D007501:Iron; D007527:Isoenzymes; D008670:Metals; D011994:Recombinant Proteins; D013869:Thiolester Hydrolases; D015032:Zinc; D000099301:Hydroxyacylglutathione Hydrolase