Rubrerythrin from Clostridium perfringens: Cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function
Rubrerythrin from Clostridium perfringens: Cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function
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DOI：
10.1128/jb.178.24.7152-7158.1996
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发表时间：
1996-12-01
影响因子：
3.2
通讯作者：
Teuber, M
中科院分区：
文献类型：
--
作者：
Lehmann, Y;Meile, L;Teuber, M
食源性病原体产气荚膜梭菌是一种专性厌氧菌，在氧化应激条件下会生长。在蛋白质提取物中，我们寻找超氧化物歧化酶（SOD）活性，它可能清除在这种应激条件下产生的剧毒超氧自由基。使用经典测定法检测产气荚膜梭菌蛋白电泳后凝胶上的 SOD 活性，我们获得了指示 SOD 活性的三个主要条带的模式。通过三个色谱步骤纯化代表最亮条带的蛋白质。根据从蛋白质N端确定的20个氨基酸，我们设计了简并寡核苷酸探针来分离相应的基因。我们最终测序了一个由 195 个氨基酸组成的开放阅读框（分子量为 21,159 Da），与脱硫弧菌红红蛋白具有很强的同源性；因此，我们假设从产气荚膜梭菌中克隆了红红蛋白基因，并将其命名为rbr。新检测到的来自产气荚膜杆菌的红红菊酯的 C 端区域含有一个特征性的非血红素、非硫铁结合位点 -Cys-X-X-Cys-(X)(12)-Cys-X-X-Cys——与来自普通产气荚膜杆菌的红红菊酯中发现的相似。此外，三个-Glu-X-X-His-序列可以代表来自产气荚膜梭菌的二铁结合。红红菊酯作为SOD的生物学功能通过缺乏SOD活性的大肠杆菌突变菌株的rbr基因的功能互补得到证实。因此，我们假设红红菊酯起到氧自由基清除剂的作用。
The food-borne pathogen Clostridium perfringens, which is an obligate anaerobe, showed growth under conditions of oxidative stress. In protein extracts we looked for superoxide dismutase (SOD) activities which might scavenge highly toxic superoxide radicals evolving under such stress conditions. Using the classical assay to detect SOD activity on gels after electrophoresis of C. perfringens proteins, we obtained a pattern of three major bands indicating SOD activity. The protein representing the brightest band was purified by three chromatographic steps. On the basis of 20 amino acids determined from the N terminus of the protein, we designed a degenerate oligonucleotide probe to isolate the corresponding gene. We finally sequenced an open reading frame of 195 amino acids (molecular mass, 21,159 Da) with a strong homology to the Desulfovibrio vulgaris rubrerythrin; therefore, we assumed to have cloned a rubrerythrin gene from C. perfringens, and we named it rbr. The C-terminal region of the newly detected rubrerythrin from C. perfringens contains a characteristic non-heme, non-sulfur iron-binding site -Cys-X-X-Cys-(X)(12)-Cys-X-X-Cys- similar to that found in rubrerythrin from D. vulgaris. In addition, three -Glu-X-X-His- sequences could represent diiron binding from C. perfringens. A biological function of rubrerythrin as SOD was confirmed with the functional complementation by the rbr gene of an E. coli mutant strain lacking SOD activity. We therefore suppose that rubrerythrin plays a role as a scavenger of oxygen radicals.