Oxidative stress response in an anaerobic hyperthermophilic archaeon: presence of a functional peroxiredoxin in Pyrococcus horikoshii.
Oxidative stress response in an anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was analyzed by two-dimensional gel electrophoresis. When P. horikoshii was grown on medium supplemented with air, a marked increase in the level of a 25-kDa protein was observed in comparison with cells grown under anaerobic conditions. The N-terminal amino acid sequence of the protein was determined to be VVIGEKFPEVEVKTTHGVIKLPDYF, which coincides with that of the putative alkyl hydroperoxide reductase that has been predicted in the genome database of P. horikoshii. The gene (PH1217) encoding the protein was cloned and expressed in Escherichia coli. The produced enzyme was a hyperthermostable peroxiredoxin whose activity was not lost after incubation at 90 degrees C for 20 min. The enzyme catalyzes the reduction of cumene hydroperoxide and hydrogen peroxide using dithiothreitol as an electron donor. Northern blot analysis revealed that the transcription of the gene increased by the addition of exogenous oxygen and by the addition of an oxidative stress-inducing reagent, and reached maximum within 30 min. These results suggest that the peroxiredoxin plays an important role in the peroxide-scavenging system in an anaerobic archaeon P. horikoshii.
Kawakami
Ryushi
R
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan.
Sakuraba
Haruhiko
H
Kamohara
Shintaro
S
Goda
Shuichiro
S
Kawarabayasi
Yutaka
Y
Ohshima
Toshihisa
T
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D030541:Databases, Genetic; D004229:Dithiothreitol / Q000494:pharmacology; D004583:Electrons; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D016678:Genome; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D054464:Peroxiredoxins; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D012313:RNA / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D019843:Archaeal Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D012313:RNA; D010544:Peroxidases; D054464:Peroxiredoxins; D010100:Oxygen; D004229:Dithiothreitol