We have previously reported that Saccharomyces cerevisiae has three glutathione peroxidase homologues (GPX1, GPX2, and GPX3) (Inoue, Y. et al., J. Biol. Chem. 274, 27002-27009, 1999). Of these, the GPX2 gene product (Gpx2) shows the greatest similarity to phospholipid hydroperoxide glutathione peroxidase. Here we show that GPX2 encodes an atypical 2-Cys peroxiredoxin, which uses thioredoxin as an electron donor. Gpx2 was essentially in a reduced form even in mutants defective in glutathione reductase or glutaredoxin under oxidative stressed conditions. On the other hand, Gpx2 was partially oxidized in a mutant defective in cytosolic thioredoxin (trx1trx2) under non-stressed conditions, and completely oxidized in tert-butyl hydroperoxide-treated cells of trx1trx2 and thioreoxin reductase-deficient mutant cells. Alanine scanning of cysteine residues of Gpx2 revealed that an intramolecular disulfide bond was formed between Cys37 and Cys83 in vivo. Gpx2 was purified to determine whether it functions as a peroxidase that uses thioredoxin as an electron donor in vitro. Gpx2 reduced H2O2 and tert-butyl hydroperoxide in the presence of thioredoxin, thioredoxin reductase and NADPH (for H2O2: Km = 20 M, kcat = 9.57 x 102 s1; for tert-butyl hydroperoxide: Km = 62.5 M, kcat = 3.68 x 102 s1); however, it showed remarkably less activity toward these peroxides in the presence of glutathione, glutathione reductase and NADPH. The sensitivity of yeast cells to tert-butyl hydroperoxide was found to be exacerbated by the co-existence of Ca2+, a tendency which was most obvious in gpx2 cells. Though the redox state of Gpx2 was not affected by Ca2+, the Gpx2 level was markedly increased in the presence of both tert-butyl hydroperoxide and Ca2+. Gpx2 is likely to play an important role in the protection of cells from oxidative stress in the presence of Ca2+.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|