Oxidative stress response in an anaerobic hyperthermophilic archaeon:: Presence of a functional peroxiredoxin in Pyrococcus horikoshii
Oxidative stress response in an anaerobic hyperthermophilic archaeon:: Presence of a functional peroxiredoxin in Pyrococcus horikoshii
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DOI：
10.1093/jb/mvh157
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发表时间：
2004-10-01
影响因子：
2.7
通讯作者：
Ohshima, T
中科院分区：
文献类型：
--
作者：
Kawakami, R;Sakuraba, H;Ohshima, T
采用双向凝胶电泳技术研究了厌氧超嗜热古菌Pyrococcus horikoshii OT-3的氧化应激反应。当P. horikoshii生长在补充有空气的培养基上时，与在厌氧条件下生长的细胞相比，观察到25-kDa蛋白质的水平显著增加。该蛋白的N-末端氨基酸序列被确定为VVIGEKFPEVEVKTTHGVIKLPDYF，这与已在堀越拟杆菌基因组数据库中预测的推定的烷基过氧化氢还原酶的序列一致。克隆了编码该蛋白的基因PH 1217，并在大肠杆菌中表达。所产生的酶是一个hyperthermostable peroxiredoxin，其活性在90 degreesC孵育20 min后没有丢失。该酶催化氢过氧化枯烯和过氧化氢的还原，使用二硫苏糖醇作为电子供体。北方印迹分析表明，该基因的转录增加，通过添加外源性氧和氧化应激诱导剂，并在30分钟内达到最大值。这些结果表明，过氧化物酶在厌氧古菌P. horikoshii的过氧化物清除系统中起着重要的作用。
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 90degreesC 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.