A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase
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- A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase
- Jung, BG; Lee, KO; Lee, SS; Chi, YH; Jang, HH; Kang, SS; Lee, K; Lim, D; Yoon, SC; Yun, DJ; Inoue, Y; Cho, Moo Je; Lee, SY
- SACCHAROMYCES-CEREVISIAE; MAMMALIAN PEROXIREDOXIN; STRESS; YEAST; PROTEINS; SELENOCYSTEINE; EXPRESSION; REDUCTASE; CLONING; CITRUS
- Issue Date
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- JOURNAL OF BIOLOGICAL CHEMISTRY, v.277, no.15, pp.12572 - 12578
- A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H2O2 and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(151)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or cys(136).
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