Characterization of novel hexadecameric thioredoxin peroxidase from Aeropyrum pernix K1

A gene (APE2278) encoding the peroxiredoxin (Prx) homologous protein of yeast and human was identified in the genome data base of the aerobic hyperthermophilic archaeon Aeropyrum pernix. We cloned the gene and produced the encoded protein in Escherichia coli cells. The isolated recombinant protein showed peroxidase activity in vitro and used the thioredoxin system of A. pernix as an electron donor. These results indicate that the recombinant protein is in fact thioredoxin peroxidase (ApTPx) of A. pernix. Immunoblot analysis revealed that the expression of ApTPx was induced as a cellular adaptation in response to the addition of exogenous H2O2 and may exert an antioxidant activity in vivo. An analysis of the ApTPx oligomers by high pressure liquid chromatography and electron microscopic studies showed that ApTPx exhibited the hexadecameric protein forming 2-fold toroid-shaped structure with outer and inner diameters of 14 and 6 nm, respectively. These results indicated that ApTPx is a novel hexadecameric protein composed of two identical octamers. Although oligomerization of individual subunits does not take place through an intersubunit-disulfide linkage involving Cys(50) and Cys(213), Cys(50) is essential for the formation of the hexadecamer. Mutagenesis studies suggest that the sulfhydryl group of Cys(50) is the site of oxidation by peroxide and that oxidized Cys(50) reacts with the sulfhydryl group of Cys(213) of another subunit to form an intermolecular disulfide bond. The resulting disulfide can then be reduced by thioredoxin. In support of this hypothesis, ApTPx mutants lacking either Cys(50) or Cys(213) showed no TPx activity, whereas the mutant lacking Cys(207) had a TPx activity. This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.