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首页> 外文期刊>Journal of bacteriology >Characterization of the PIB-Type ATPases Present in Thermus thermophilus
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Characterization of the PIB-Type ATPases Present in Thermus thermophilus

机译：嗜热栖热菌中PIB型ATP酶的表征

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PIB-type ATPases transport heavy metals (Cu2+, Cu+, Ag+, Zn2+, Cd2+, Co2+) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative PIB-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn2+/Cd2+-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu+, while CopB was found to have greater activity in the presence of Cu2+. The putative Zn2+/Cd2+-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn2+/Cd2+-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn2+ and Cd2+ as well as by Cu+. Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.
机译：P IB 型ATPase转运重金属（Cu 2 + ，Cu + ，Ag + ，Zn 2 + ，Cd 2 + ，Co 2 + ），在这些金属的稳态和生物耐受机制中起着关键作用。嗜热栖热菌基因组（HB8和HB27）中存在三种编码假定的P IB 型ATPase的基因：TTC1358，TTC1371和TTC0354基因;这些基因分别标注为两个铜转运蛋白（CopA和CopB）基因和一个锌镉转运蛋白（Zn 2 + / Cd 2 + -ATPase）基因。我们使用嗜热链球菌表达系统克隆并表达了具有8His标签的三种蛋白质。纯化后，每种蛋白质在ATP和 p -硝基苯基磷酸酯（ p NPP）为底物的情况下均显示在65°C具有磷酸二酯酶活性。发现CopA在Cu + 存在下具有更高的活性，而CopB在Cu 2 + 存在下具有更高的活性。推测的Zn 2 + / Cd 2 + -ATPase在N末端被截短，令人惊讶的是，铜在体外被激活，但没有被铜激活。通过锌或镉。然而，当在大肠杆菌中表达时，假定的Zn 2 + / Cd 2 + -ATPase可以作为全长蛋白被分离出来，并且通过Zn 2 + 和Cd 2 + 以及Cu + 的添加。构建其中三个P型ATP酶各自被缺失的突变株。在每种情况下，缺失增加了菌株在培养基中存在铜的情况下对生长的敏感性，表明这三者均可将铜泵出细胞并在铜的解毒中起作用。

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