Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum.
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.
Bernier-Villamor
Laura
L
Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008, Granada, Spain.
Navarro
Eusebio
E
Sevilla
Francisca
F
Lázaro
Juan-José
JJ
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D007700:Kinetics; D018532:Pisum sativum / Q000201:enzymology / Q000235:genetics*; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism; D054464:Peroxiredoxins; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D018744:DNA, Plant; D011994:Recombinant Proteins; D010544:Peroxidases; D054464:Peroxiredoxins