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Structural studies on a mitochondrial glyoxalase II. J Biol Chem 2005 Dec 09;280(49):40668-75

Date

10/18/2005

Pubmed ID

16227621

Pubmed Central ID

PMC1343529

DOI

10.1074/jbc.M509748200

Scopus ID

2-s2.0-28844503390 (requires institutional sign-in at Scopus site)   76 Citations

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).

Author List

Marasinghe GP, Sander IM, Bennett B, Periyannan G, Yang KW, Makaroff CA, Crowder MW

Author

Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette University




MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Arabidopsis
Binding Sites
Cloning, Molecular
Crystallography, X-Ray
Electron Spin Resonance Spectroscopy
Gene Expression
Humans
Iron
Isoenzymes
Kinetics
Magnetic Resonance Spectroscopy
Metals
Mitochondria
Models, Molecular
Molecular Sequence Data
Molecular Weight
Polymerase Chain Reaction
Recombinant Proteins
Substrate Specificity
Thiolester Hydrolases
Zinc