胃幽門螺旋桿菌是ㄧ種微需氧的革蘭氏陰性菌，會造成胃潰瘍社甚至是胃癌，輔酶A對生物體來說是ㄧ個重要的輔因子，因為他參予了許多重要的代謝反應，而磷酸泛酸醯基乙胺腺苷轉移酶在輔酶A生合成途徑中是速率限制步驟，因此我們選擇磷酸泛酸醯基乙胺腺苷轉移酶作為我們研究的目標蛋白。
為了瞭解磷酸泛酸醯基乙胺腺苷轉移酶催化的過程，我們解出了磷酸泛酸醯基乙胺腺苷轉移酶與其配體結合的復合體結構，包括與三磷酸腺苷的，與磷酸泛酸硫氫乙胺的，以及與輔酶A的，並與沒結合配體的磷酸泛酸醯基乙胺腺苷轉移酶做比較，來找出在催化過程中，哪些重要的胺基酸與配體有交互作用。
我們也隨機突變了胃幽門桿菌基因序列上的基因，並發現其中一種較為特殊的突變株 : I4V/N76Y，它不像一般的磷酸泛酸醯基乙胺腺苷轉移酶為同源六聚體結構，I4V/N76Y反而是ㄧ種有區域相互結合特性的四聚體結構。我們也比較了此突變型與正常型光譜學,酵素動力學集結構上的差異。
另外，我們利用了以結構為基礎的高通量篩選技術篩選出一些化合物，並找出其中可以抑制磷酸泛酸醯基乙胺腺苷轉移酶活性且有潛力成為胃幽門螺旋菌抑制劑的化合物，這可能會對未來在對抗胃幽門螺旋菌的藥物設計上提供了一些有價值的訊息。

Helicobacter pylori (H. pylori) is a gram-negative microaerophilic bacterium that causes chronic gastritis and stomach cancer. Coenzyme A (CoA) is an essential cofactor in all living organisms, which participates in numerous metabolic pathways. For this reason, we choose phosphopantetheine adenylytransferase (PPAT) as a target protein in our research, which is the rate-limiting enzyme in CoA biosynthesis.
To investigate the PPAT catalytic mechanism, complex structures of H. pylori PPAT with other ligands, including ATP, phosphopantetheine, and CoA have been determined. Structural comparisons between apo-form PPAT and complex form have been performed and identified some critical residues interacting with these ligands.
We randomly mutated the residues in the Helicobacter pylori PPAT sequence and described the crystal structure of one of these mutants (I4V/N76Y). Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. These two structures between wild-type PPAT and I4V/N76Y PPAT have been characterized by circular dichroism, enzyme kinetics, and crystallography approaches.
In addition, structure based high-throughput screen has been employed and some compounds have been utilized to examine the potent inhibitor to inhibit H. pylori PPAT activity. This may provide valuable information for drug design to against the H. pylori infection in the future.

Abstract I
中文摘要 II
Content III
List of figures VI
List of tables IX
1. Introduction 1
1.1 Helicobacter pylori 1
1.2 Coenzyme A 2
1.3 Phosphopantetheine adenylyltransferase 3
1.4 Domain swapping – I4V/N76Y 5
1.5 Discover novel inhibitors 6
1.6 Aim of the study 7
2. Materials and methods 9
2.1 Materials 9
2.2 Over expression of H. pylori 10
2.3 Purification of H. pylori and its mutants 11
2.4 H. pylori PPAT crystallization 12
2.5 X-ray diffraction data collection 12
2.6 Molecular replacement and crystallographic refinement 13
2.7 Circular dichroism spectroscopy 14
2.8 Pyrophosphate assay 15
2.9 Kinetic analysis of forward reaction 16
2.10 Analytical ultracentrifuge 17
2.11 UV-visible absorption spectroscopy 17
2.12 Virtual high-throughput screening (vHTS) 18
2.13 Quantitative H. pylori inhibition assay 18
2.14 Steady-state kinetic inhibition assay using D-amethopterin 19
2.15 Isothermal titration calorimetry (ITC) 20
3. Result and discussion 21
3.1 Protein purification 21
3.2 Structural insight into substrate binding mode of wild-type HpPPAT 21
3.2.1 Crystal structure of apo-form PPAT and complex form PPAT 21
3.2.2 ATP binding to PPAT 22
3.2.3 Ppant binding to PPAT…………………………………………………23
3.2.4 HpPPAT catalytic process 23
3.3 I4V/N76Y-domain swapping conformation 24
3.3.1 Crystal structure of I4V/N756Y-CoA and I4V/N76Y-ATP complex 24
3.3.2 Circular dichroism spectroscopy 26
3.3.3 Kinetic analysis of I4V/N76Y 26
3.3.4 Asparagine 76 mutants 27

3.4 Discover a novel inhibitors of H. pylori PPAT 29
3.4.1 Virtual high-throughput screening 29
3.4.2 H. pylori inhibition assay 30
3.4.3 Steady-state inhibition assay 31
3.4.4 ITC 31
3.4.5 Binding model 32
4. Conclusion 34
5. Figures 36
6. Tables 71
7. Reference 77

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