Poster: Oxidative stress

Abs # 81: Molecular characterization of a novel plant thioredoxin-dependent peroxidase a that has a high sequence identity to PHGPx

Presenter:	Park, Soo Kwon , sookwonpark@hotmail.com
Authors	Park, Soo Kwon  (A) (C)  Jeon, Min Gyu  (A)   Lee, Seung Sik  (A) (C)  Chi, Yong Hun  (A) (C)  Jang, Ho Hee  (A) (C)  Cho, Moo Je  (B) (C)  Lee, Sang Yeol  (A) (B)
Affiliations:	(A): Environmental Biotechnology Research Center (B): Division of Applied Life Science (BK21 Program) (C): Plant Molecular Biology and Biotechnology Research Center

A cDNA, C3C-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases and specifically expressed in flowers was isolated from a Chinese cabbage cDNA library. C3C-TPx encodes a preprotein of 232 amino acids containing a putative chloroplast targeting sequence at the N-terminus and three highly conserved Cys residues (Cys107, Cys136 and Cys155). The mature form of enzyme without the signal peptide was expressed in E. coli, and the recombinant protein was found to utilize thioredoxin (Trx) as an electron donor. In the presence of a Trx system, the protein efficiently reduces H2O2 and organic hydroperoxides but not phospholipid hydroxyperoxide. Complementation analysis shows that overexpression of the C3C-TPx protein restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the in vivo antioxidant activity of C3C-TPx requires the electrons donated from Trx, but not from GSH. Mutation of the three Cys residues into Ser shows that all three conserved Cys residues are required for Trx-dependent peroxidase (TPx) activity and that Cys107 is the primary site of oxidation by peroxide. Unlike yeast 2Cys-TPx, C3C-TPx does not form intermolecular disulfide bonds, as shown by nonreducing SDS/PAGE analysis. Tryptic peptide analysis by matrix-assisted laser desorption/ionization time of flight mass spectrometry shows that Cys155 can form a disulfide bond with either Cys107 or Cys136. Based on these observations, we propose a novel enzymatic mechanism for C3C-TPx that involves all three Cys residues in the reaction cycle. [This work was supported by a grant from the Korea Science and Engineering Foundation (KOSEF) to the Environmental Biotechnology Research Center]