Molecular and biochemical characterisation of a novel type II peroxiredoxin (XvPrx2) from the resurrection plant Xerophyta viscosa

A type II peroxiredoxin gene (XvPrx2) was isolated from a Xerophyta viscosa (Baker) cDNA cold-stress library. The polypeptide displayed significant similarity to other plant type II peroxiredoxins, with the conserved amino acid motif (PGAFTPTCS) proposed to constitute the active site of the enzyme. Northern blot analyses showed that XvPrx2 gene was stress-inducible in response to abiotic stresses while gel analyses revealed that XvPrx2 homologues exist within the X. viscosa proteome. Using a yellow fluorescent reporter protein, the XvPrx2 protein localised to the cytosol. A mutated protein (XvV7) was generated by converting the valine at position 76 to a cysteine and an in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV7, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred to Escherichia coli cells overexpressing either XvPrx2 or XvV7. The XvPrx2 activity was maximal with DTT as electron donor and H2O2 as substrate. Using E. coli thioredoxin, a 2-15-fold lower enzyme activity was observed. The XvPrx2 activity with glutathione was significantly lower and glutaredoxin had no measurable effect on this reaction. The XvV7 protein displayed significantly lower activity compared with XvPrx2 for all substrates assessed.