Co-Authors:
Faltin, Z., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Camoin, L., Inst. Conchin de Génétique Moléculaire, CNRS UPR 415, 22 Rue Méchain, F-75014 Paris, France
Ben-Hayyim, G., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Perl, A., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Beeor-Tzahar, T., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Strosberg, A.D., Inst. Conchin de Génétique Moléculaire, CNRS UPR 415, 22 Rue Méchain, F-75014 Paris, France
Holland, D., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Eshdat, Y., Dept. of Fruit Tree Breeding and Molecular Genetics, Agricultural Research Organization, P.O. Box 6, 50250 Bet-Dagan, Israel
Abstract:
A citrus salt-stress associated protein (Cit-SAP), partially purified from citrus cultured cells, was previously identified as the first plant phospholipid hydroperoxide glutathione peroxidase (PHGPx). The nucleotide sequence of its isolated gene (csa) revealed that a TGT, known as codon for Cys, encodes the presumed catalytic residue 41 in the polypeptide chain of Cit-SAP. In animals, a TGA encodes the rare amino acid selenocysteine (Sec) as the catalytic residue of the analogous enzyme. It is of interest to establish whether the TGT codon for this catalytic residue in the plant enzyme is indeed translated to Cys and not to Sec, and to demonstrate the effect of such a change, if it exists, on the nature of the enzymatic activity of the plant enzyme as compared to that of the animal. In the present study, we have purified for the first time, by affinity chromatography, enzymatically active citrus PHGPx from recombinant Escherichia coli bearing the csa gene. Tryptic digestion of the purified enzyme followed by HPLC afforded the isolation of a peptide which contains residue 41, and its sequence analysis revealed that this residue is indeed a Cys, and not Sec. The enzymatic activity and specificity of the recombinant Cit-SAP was found to be similar to that observed before for the partially purified plant enzyme. However, the rate of this activity was much lower towards phospholipid hydroperoxides, and none towards hydrogen peroxide, as compared to that of the animal analogue. It is therefore suggested that the presence of a Cys, and not Sec, as the catalytic residue in the plant enzyme, affects its enzymatic activity and may determine a different biological role for the plant PHGPx from that of the animal.
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