A Chinese Cabbage cDNA with High Sequence Identity to Phospholipid Hydroperoxide Glutathione Peroxidases Encodes a Novel Isoform of Thioredoxin-dependent Peroxidase

A Chinese Cabbage cDNA with High Sequence Identity to Phospholipid Hydroperoxide Glutathione Peroxidases Encodes a Novel Isoform of Thioredoxin-dependent Peroxidase

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...

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Published in:The Journal of biological chemistry Vol. 277; no. 15; pp. 12572 - 12578
Main Authors: Jung, Bae Gyo, Lee, Kyun Oh, Lee, Seung Sik, Chi, Yong Hun, Jang, Ho Hee, Kang, Soon Suk, Lee, Kyunghee, Lim, Dongbin, Yoon, Sung Chul, Yun, Dae-Jin, Inoue, Yashiharu, Cho, Moo Je, Lee, Sang Yeol
Format: Journal Article
Language:English
Published: United States Elsevier Inc 12-04-2002
American Society for Biochemistry and Molecular Biology
Subjects:
Amino Acid Sequence
Brassica
Brassica - genetics
Catalysis
Cloning, Molecular
Disulfides - metabolism
DNA, Complementary
Glutathione - metabolism
Humans
Hydrogen Peroxide - pharmacology
Molecular Sequence Data
Mutagenesis, Site-Directed
Neoplasm Proteins
Peroxidases - chemistry
Peroxidases - genetics
Peroxidases - metabolism
Peroxiredoxin III
Peroxiredoxins
PHCC-Tpx gene
Phylogeny
Sequence Homology, Amino Acid
Substrate Specificity
Thioredoxins - metabolism
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Summary: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 (Cys107, Cys136, and Cys155). 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 Cys107 is the primary attacking site by peroxide, and oxidized Cys107 reacts with Cys155-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 Cys155 can form a disulfide bond with either Cys107 or Cys136.
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content type line 23
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M110791200