Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum

J Exp Bot. 2004 Oct;55(406):2191-9. doi: 10.1093/jxb/erh238. Epub 2004 Aug 27.


A cDNA sequence coding for a pea (Pisum sativum L.) 2-Cys peroxiredoxin (2-Cys Prx) has been cloned. The deduced amino acid sequence showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%), Arabidopsis thaliana (75%), and Spinacia oleracea (75%), and contained a chloroplast target sequence at its N-terminus. The mature enzyme, without the transit peptide, has a molecular mass of 22 kDa as well as two cysteine residues (Cys-53 and Cys-175) which are well conserved among proteins of this group. The protein was expressed in a heterologous system using the expression vector pET3d, and was purified to homogeneity by three sequential chromatographic steps. The enzyme exhibits peroxidase activity on hydrogen peroxide (H(2)O(2)) and t-butyl hydroperoxide (TBHP) with DTT as reducing agent. Although both pea Trxs f and m reduce oxidized 2-Cys Prx, Trx m is more efficient. The precise conditions for oligomerization of 2-Cys Prx through extensive gel filtration studies are also reported. The transition dimer-decamer produced in vitro between pH 7.5 and 8.0 and the influence of DTT suggest that a great change in the enzyme quaternary structure of 2-Cys Prx may take place in the chloroplast during the dark-light transition. In addition, the cyclophilin-dependent reduction of chloroplast 2-Cys Prx is shown.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • DNA, Plant / genetics
  • Kinetics
  • Peas / enzymology
  • Peas / genetics*
  • Peroxidases / genetics*
  • Peroxidases / metabolism
  • Peroxiredoxins
  • Recombinant Proteins / metabolism


  • DNA Primers
  • DNA, Plant
  • Recombinant Proteins
  • Peroxidases
  • Peroxiredoxins