在金黃葡萄球菌質體pI258上的重金屬鎘抗性基因，早於1992年已被證實具有轉運鎘的作用，之後由於它的結構特殊性，因此將此種抗鎘幫浦、與其他類似的酵素，共同歸類為CPx-ATPases的酵素家族。然而，目前對於其排除鎘的酵素機制仍不清楚。因此本論文藉著測量CadA-ATPase水解ATP的酵素活性，針對具有高度保留特性的胺基酸序列如:Cys-X-X-Cys、及Cys-Pro-Cys進行探討，並且期盼能建立起一套完整的酵素活性監測方法，以因應未來進一步純化蛋白的工作所需。雖然對於胺基酸序列Cys-X-X-Cys，在CPx-ATPases所扮演的角色，目前尚無定論。但一般相信，它可能會結合金屬離子，接著利用分解ATP所產生的能量，透過CPC所形成的通道，將金屬離子排出細胞外。因此含有CadA的細菌，便可存活在高濃度鎘的環境之中。在本研究中，藉由基因工程技術將原有Cys-X-X-Cys、及Cys-Pro-Cys上的Cysteine胺基酸，改變為Serine、或Glycine，所產生的突變基因，轉移至pKJ100 表達系統中，並在cadmium-sensitive的大腸桿菌RW3110中表達。當這些大腸桿菌，在生長時分別將重金屬鎘、鋅、以及鉛加入培養基中，發現除了部分Cys-X-X-Cys 的突變株，保有些許重金屬抗性之外，其他的生長狀況都比野生株CadA還差。然而透過Enzyme coupling assay，來分析這些突變種蛋白，在水解ATP的酵素活性的差異，結果卻發現這些突變蛋白並不具備水解ATP酵素活性。同時為了得到高純度的CadA蛋白，我們嘗試利用界面活性劑Triton X-100來純化，卻因為Enzyme coupling assay的干擾作用，而無法得到良好的再現性。除此之外，經由in vitro translation所合成的CadA蛋白也同樣對於ATPase assay有干擾作用。由於大腸桿菌細胞膜中含有許多不同於CadA的ATPase，利用剔除F0F1-ATPase、Kdp-ATPase、ZntA的大腸桿菌突變株BF2000，來進一步探討可能扮演直接轉運金屬離子的Cys-Pro-Cys。在抗鎘的表現分析之中，我們發現ATP-binding domain的突變以及所有的Cys-Pro-Cys突變都失去抗性。除此之外，在微量的鎘存在之下CadA的酵素活性能約略增加，但更高濃度的鎘卻無法得到更強的酵素活性。實驗中我們也發現P-ATPase 的抑制劑，如Vanadate 似乎不能影響CadA的酵素活性。最後，以Phosphate precipitation method與 GST-CadA 蛋白表達系統，建立一套較為可行的酵素活性監測方法，以便做為未來純化CadA蛋白時的監測所需。

The cadA gene encoded cadmium resistance found in staphylococcal plasmid pI258 has been characterized previously as a cadmium-efflux P-ATPase. Recently, based on its unique structural features, the CadA cadmium resistance ATPase, alone with other similar membrane efflux ATPases were further classified as a member of CPx-ATPases, these features include a conserved CPx motif and a CxxC motif. However, without direct evidence, the exact role of CxxC and CPx motif in cadmium translocation mechanism remain undefined. In this thesis study, the two conserved unique motifs were studied their role in cadmium resistances as well as ATP hydrolysis. To determine the role of cysteine residues within these motifs, eight cysteine mutants were prepared and expressed them in a cadmium-sensitive E. coli strain RW3110. Surprisingly, some cysteine mutants of CxxC motif seem less sensitive to heavy metal than those cysteine mutants in CPC motif. On the other hand, ATPase assay using enzyme coupling assay was established to monitored the ATP hydrolytic activity of those cysteine mutants. All cysteine mutants have shown various and reduced activity compared to wild type CadA, especially the activities from some mutants of CxxC motif were less active than those of CPC motif. Suggesting that the C26, and possibly the C23 residue may participate in other role in CadA enzyme cycle, instead of directly involving in ATP-dependent metal translocation process. Meanwhile, to eliminate the possible interference in membrane vesicles, Triton X-100-solubilized or in vitro translational CadA were prepared for ATPase assays in this study. However, both of these methods did not generate a convinced result. More recently, an E. coli strain with triple mutation (unc-, kdp-, zntA-) and alone with a new CadA expression system were included in this study. Using this latest CadA expression system, we found that b-mercaptoethanol was able to slightly increase the ATPase activity of CadA. Even though the activity of CadA achieved in this study was less than other P-ATPases, our data did create a new avenue toward the better understandings of CadA enzyme mechanism and provide a new direction for future protein studies of CadA and other similar CPx-ATPases.

Table of Contents……………………………………………………. i
List of Tables………………………………………………………… iii
List of Figures……………………………………………………….. iv
Chapter 1. Introduction…………………………………………….. 1
1.1 Bacterial resistance to toxic metals…………………………… 1
1.2 Cadmium, one of the toxic components found in the environment…………………………………………………… 1
1.3 Introduction to bacterial cadmium resistance………………….. 2
1.4 The structure of cad operon……………………………………. 4
1.5 The role of CadA as an energy-dependent extruder……………. 5
1.6 The CadA, the metal translocating P-type ATPases……………. 6
1.6.1 CadA belongs to the P-type ATPase superfamily…………. 6
1.6.2 P-type ATPase superfamily……………………………….. 7
1.6.3 CadA is a member of CPx-type ATPases…………………. 8
1.6.4 Topological structures and conserved regions of P-type ATPases…………………………………………………….. 9
1.6.5 The CXXC and CPC motifs in CadA……………………... 10
1.6.6 The mechanism of heavy metal transduction of P-ATPase... 12
1.7 Rationale and experimental approach………………………….. 14
Chapter 2. Materials and methods………………………………… 15
2.1 Materials………………………………………………………... 15
2.2 Bacterial strain and plasmid…………………………………… 15
2.3 Cadmium resistance assay…………….…………………….…. 15
2.4 Preparation of membrane vesicles…………………………….. 17
2.5 In vitro transcription/translation……………………………….. 18
2.6 ATPase assay (coupling method)……………………………… 18
2.7 ATPase assay (Phosphate precipitation method)………………. 19
2.8 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) andWestern blotting……………………………………………….. 19
Chapter 3. Results…………………………………………………… 21
3.1 Mutagenesis of pGST-cadA…………………………………… 21
3.2 Cadmium sensitivity of CadA mutants in RW3110 cells……… 22
3.3 ATPase assay of CadA mutants in RW3110 cells by coupling method…………………………………………………………. 23
3.4 Cadmium sensitivity and ATPase activity of CadA mutants inBF2000 cells…………………………………………………… 25
Chapter 4. Discussion……………………………………………….. 44
References…………………………………………………………… 52

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