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 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 H(2)O(2) 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(155)-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).
Jung
Bae Gyo
BG
Division of Applied Life Sciences (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
Lee
Kyun Oh
KO
Lee
Seung Sik
SS
Chi
Yong Hun
YH
Jang
Ho Hee
HH
Kang
Soon Suk
SS
Lee
Kyunghee
K
Lim
Dongbin
D
Yoon
Sung Chul
SC
Yun
Dae-Jin
DJ
Inoue
Yashiharu
Y
Cho
Moo Je
MJ
Lee
Sang Yeol
SY
D000595:Amino Acid Sequence; D001937:Brassica / Q000235:genetics*; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D061105:Peroxiredoxin III; D054464:Peroxiredoxins; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism
D018076:DNA, Complementary; D004220:Disulfides; D009363:Neoplasm Proteins; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D010544:Peroxidases; C516115:PRDX3 protein, human; D061105:Peroxiredoxin III; D054464:Peroxiredoxins; D005978:Glutathione