Purification and biochemical characterization of LeTPx. A glutathione-dependent thioredoxin peroxidase

A tomato cDNA clone (LeTPx), encoding a nucleic acid homologue of thioredoxin peroxidase, was isolated as a BI-GST interactor using a yeast two-hybrid screen. Expression of LeTPx protected yeast cells from peroxide (t-butylhydroperoxide, cumene hydroperoxide, and H2O2)-induced cell death. Co-expression of LeTPx with Bax did not prevent cell death (N. Atanassova, M.Sc. thesis, MAICh). To investigate the means by which LeTPx was capable of conferring such qualities to cells undergoing oxidative stress, LeTPx was expressed in E. coli and purified by Ni-NTA agarose affinity chromatography. Also, the other prptyein components (Trxh, NTR), which are considered to be essential for thioredoxin-dependent peroxidase activity, were expressed and purified. Purified LeTPx was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalysed oxidation system. Unlike other peroxiredoxins, LeTPx showed glutathione-dependent thioredoxin peroxidase activity. The specific activity of LeTPx in this reaction was significantly dependent on the concentration of GSH. The peroxidase activity of LeTPx towards t-butyl hydroperoxide and cumene hydroperoxide was approximately similar to that with H2O2. Compared to other peroxiredoxins, the LeTPx protein demonstrated specific activity of about 100 times lower than that of a highly homologous protein from Poplar sieve tubes. Previous studies with peroxiredoxins have shown that peroxiredoxins did not possess glutathione peroxidase (GPx) activity. in contrast to the other peroxiredoxin enzymes, LeTPx exhibited Gpx activity. The GPx activity of LeTPX was found to be close to its peroxidase activity. As was the case with the thioredoxin peroxidese activity, the GPX activity of LeTPX was highly dependent on the concentration of GSH. These data indicate that the catalytic activity of LeTPx is significantly dependent on GSH. LeTPx is the only peroxiredoxin reported so far to use GSH in the thioredoxin dependent system. However, the mechanism of the participation of GSH in the peroxidase activity remains unknown.