title: A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase.
abstract: 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).
authors.affiliation: Division of Applied Life Sciences (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
authors.forename: Bae Gyo
authors.initials: BG
authors.lastname: Jung
authors.forename: Kyun Oh
authors.initials: KO
authors.lastname: Lee
authors.forename: Seung Sik
authors.initials: SS
authors.lastname: Lee
authors.forename: Yong Hun
authors.initials: YH
authors.lastname: Chi
authors.forename: Ho Hee
authors.initials: HH
authors.lastname: Jang
authors.forename: Soon Suk
authors.initials: SS
authors.lastname: Kang
authors.forename: Kyunghee
authors.initials: K
authors.lastname: Lee
authors.forename: Dongbin
authors.initials: D
authors.lastname: Lim
authors.forename: Sung Chul
authors.initials: SC
authors.lastname: Yoon
authors.forename: Dae-Jin
authors.initials: DJ
authors.lastname: Yun
authors.forename: Yashiharu
authors.initials: Y
authors.lastname: Inoue
authors.forename: Moo Je
authors.initials: MJ
authors.lastname: Cho
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
mesh_terms: 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
chemical_list: 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