Abstract
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 Cys50 and Cys213, Cys50 is essential for the formation of the hexadecamer. Mutagenesis studies suggest that the sulfhydryl group of Cys50 is the site of oxidation by peroxide and that oxidized Cys50 reacts with the sulfhydryl group of Cys213 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 Cys50 or Cys213 showed no TPx activity, whereas the mutant lacking Cys207 had a TPx activity. This is the first report on the biochemical and structural features of a novel hexadecameric thioredoxin peroxidase from the archaea.
Highlights
Reactive oxygen species (ROS)1 are generated by the incomplete reduction of oxygen during respiration or by exposure to external factors such as light, radiation, redox-cycling drugs, and stimulated host phagocytes [1, 2]
The Prx family can be divided into two subgroups as follows: 1) 2-Cys Prx proteins, which contain Cys residues at both the N- and C-terminal regions, and 2) 1-Cys Prx proteins, which contain only one Cys at the N-terminal region [23]. 1-Cys Prxs are able to reduce peroxides using DTT as electron donor, but the physiological electron donor for 1-Cys Prxs is currently unknown
It was reported that yeast contains a 1-Cys peroxiredoxin (Prx1p) with thioredoxin peroxidases (TPx) activity [17]
Summary
Construction and Expression of the Recombinant Protein—Chromosomal DNA of A. pernix K1 was prepared as described by Sako et al [15]. Protection assays were performed in 25-l reaction mixtures containing 50 mM Hepes-NaOH, pH 7.4, 5 g of GS, 3 M FeCl3, 10 mM DTT, and various concentrations of ApTPx. After 10 min at 37 °C, the remaining GS activity was measured by the addition of 2 ml of ␥-glutamyltransferase assay mixture as described previously [21]. Electron Microscopy—The purified wild type and mutant ApTPx were diluted with 50 mM Tris-HCl, pH 7.5, to final protein concentrations of 50 g/ml. The antisera were examined for specificity by an enzyme-linked immunosorbent assay and confirmed that anti-ApTPx detected ApTPx. The cells from 10-h (exponential phase) cell cultures of A. pernix were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 15 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100) and disrupted by sonication for 1 min at 4 °C. The bands were visualized by adding nitro blue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate (Bio-Rad)
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