利用雜交蛋白探討CadA和CopA兩個CPx-type ATPase的研究

Chimera studies of two CPx-type ATPases, CadA and CopA

廖志偉(Chih-Wei Liao)

指導教授 ： 蔡淦仁 ； 楊順發

中山醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

金黃色葡萄球菌產生的抗鎘能力，主要是因為細菌所攜帶的pI258質體上，擁有cadA基因所產生的CadA蛋白，由這個蛋白負責將鎘離子排出菌體外，因而產生抗鎘抗性的主要原因。過去研究發現，CadA蛋白是屬於CPx-type ATPases中的一員，具有相當類似的結構與保留相當多的氨基酸序列，並廣泛出現在其他不同受質的運輸蛋白上。為了瞭解這些結構類似的CPx-type ATPases的功能性區域，是如何辨識不同的受質，而執行不同的運輸任務。因此在本研究中，就是採用兩種不同的CPx-type ATPases進行chimera的研究，利用交換具有不同受質的CPx-type ATPases的功能性區域，用來找出CPx-type ATPases蛋白是如何辨識受質。在本篇研究中，利用E. coli中銅離子運輸蛋白copA，與金黃色葡萄球菌的cadA，進行各種不同片段的融合，而產生一系列相關的CopA/CadA和CadA/CopA chimera蛋白。在本篇研究中，我們分析了這些chimera蛋白在細菌的抗性能力，以及生化的特性。由結果顯示，N端(CXXC)n 被置換掉的chimera蛋白會失去抗性功能。可能是N端(CXXC)n是受質開始被辨識，然後活化CPC motif進而啟動金屬離子的轉移所必需的序列。然而，這些改變是否造成chimera蛋白的生化活性受到影響，目前仍在探討中。

關鍵字

雜交蛋白

並列摘要

Staphylococcal cadmium resistance mediated by penicillinase plasmid pI258 has been characterized and a CadA cadmium- transporting ATPase, a member of CPx-type ATPases, is identified to be responsible for the resistance. Recently, CPx-type ATPases with different substrates were extensively studied, and conserved structural and functional similarities among all these ATPase enzymes were noticed. In order to understand and identify the functional domains of CPx-type ATPases, especially those of the substrate recognition, as well as certain unique conserved stretches of sequences, a chimera study of using two different CPx-type ATPases were conducted. In this study, an E. coli chromosomal copper resistance gene, copA, were partially fused with the cadmium resistance gene cadA to prepare a series of chimera CopA/CadA and CadA/CopA proteins, and to determine their resistance phenotypes. Our preliminary data have shown that unless the last five C-terminal transmembrane domains from either CadA or CopA are intact in order to preserve their resistances to cadmium or copper. It is suggested that the distinct N-terminal (C-X-X-C)n domain might be essential for the initial substrate recognition to activate the conserved CPC motif of all CPx-type ATPases to turn on the metal-translocation action. However, two other major characteristic functions, the metal-dependent ATPase as well as phosphorylation are not determined yet, and these experiments are currently undertaken.

並列關鍵字

Chimera protein ； CadA ； CopA ； CPx-type ATPase

參考文獻

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