胃幽門螺旋桿菌屬格蘭氏陰性之微需氧菌，為感染胃黏膜並導致胃炎及胃潰瘍之人類病原體，可在大部分菌體所無法容忍的胃部酸性環境下存活，也是胃癌與胃黏膜相關淋巴組織淋巴瘤的重要致病因子。胃幽門螺旋桿菌造成疾病的機制尚未完全被了解，且由於所需的培養條件較一般細菌嚴苛，對基因轉殖易產生抗性，使得適當之研究系統不易建立。隨著胃幽門螺旋桿菌基因體的解碼，我們可以較方便地透過重組蛋白質的建構，來研究此一病原體。
胃幽門螺旋桿菌 26695的 HP0832 (speE) 基因被預測可轉譯為亞精胺合成酶 (spermidine synthase)。亞精胺合成酶屬於多胺分子 (polyamine) 生合成途徑所需的酵素之一，主要催化腐胺 (putrescine) 及 decarboxylated S-adenosylmethionine (dcSAM) 合成亞精胺 (spermidine)，而 dcSAM 的作用即為提供反應所需之丙胺基 (aminopropyl group)。胃幽門螺旋桿菌亞精胺合成酶的蛋白質序列與哺乳類，植物，以及其他菌種的亞精胺合成酶的序列一致性 (sequence identity) 低於 20%。本研究中，我們將 HP0832 基因 (786 bp) 建構在 pQE30 質體中，使其在大腸桿菌 SG13009 中過量表現，以取得胺基端 (N-terminus) 帶有六個組氨酸 (histidine) 的重組蛋白。HP0832 重組蛋白分子量為 31.9 kDa，以鎳螯合親和層析法 (Ni-NTA affinity chromatography) 純化的產率為 15 mg/L 菌液。將 HP0832 重組蛋白與腐胺及 dcSAM 作用可觀察到亞精胺的產生，由此確立其活性。此外，利用 HP0832 重組蛋白之抗體血清可偵測到胃幽門螺旋桿菌內生之亞精胺合成酶。胃幽門螺旋桿菌 26695 所含有之腐胺及亞精胺的莫耳比為 1:3，未發現其他多胺分子如精胺 (spermine) 及 norspermidine，由此推論亞精胺之合成途徑為提供胃幽門螺旋桿菌之多胺分子的主要來源。由於胃幽門螺旋桿菌的亞精胺合成酶缺少完整的 gatekeeping loop 序列，可能是導致其活性不穩定的原因之一，我們亦針對含有完整 gatekeeping loop 的大腸桿菌亞精胺合成酶 (EcSPDS) 做點突變之研究，以探討此一序列對亞精胺合成酶催化反應的重要性。
胃幽門螺旋桿菌 26695 的焦磷酸水解酶 (inorganic pyrophosphatase) 由 HP0620 (ppa) 基因所轉譯，屬於 family I 焦磷酸水解酶，由六個相同分子量 (20 kDa) 的單體構成六聚體 (homohexamer)，需要鎂離子作為催化反應之輔助因子 (cofactor)。胃幽門螺旋桿菌焦磷酸水解酶 (HpPPase) 水解焦磷酸 (PPi) 的反應遵循 Michaelis-Menten 酵素動力論，在最適反應 pH 値 8.0 下，kcat 和 Km 分別為 344 s-1 及 83 μM。HpPPase 可同時被含硫基及不含硫基之還原劑所活化，不同於早期研究所提出之與雙硫鍵形成相關之抑制及再活化機制。HpPPase 中唯一的半胱氨酸 Cys15 既不屬於活化中心，亦不具有演化保守性 (evolutionarily conserved)，但將此胺基酸取代後，卻導致 50% 的活性下降，以及對還原劑與氧化態麩胺基硫 (oxidized glutathione) 的敏感度降低。此外，將 Cys15 取代為絲氨酸 (serine) 亦造成熱穩定性的破壞，使 C15S HpPPase 的熱穩定性明顯低於取代活化中心之熱不穩定焦磷酸水解酶如 Y139F HpPPase 及大腸桿菌之熱不穩定焦磷酸水解酶。雖然 Cys15 並沒有位在次體介面 (subunit interface)，但研究結果顯示 C15S HpPPase 熱穩定性的下降是透過破壞兩個三聚體的介面作用 (trimer-trimer interactions) 所造成。本研究首度提出 HpPPase 唯一的半胱氨酸與焦磷酸水解酶的活化，熱穩定性，以及四級結構 (quaternary structure) 的穩定有關。
Soj 與 Spo0J 兩個蛋白質加上一個或多個 parS 序列所構成的 parABS系統，在許多細菌的染色體核質分離與細胞週期的運轉扮演關鍵性的角色。在胃幽門螺旋桿菌 26695 的圓形染色體中，以序列比對方式所得到的假設性複製起點 (replication origin) oriC 序列位在 1,609,162 bp，而 Soj (HP1139) 和一帶有 Spo0J 或 ParB 保留區 (conserved domain) 的基因 (HP1138)，以及與枯草桿菌之 parS 序列比對所得到的兩個胃幽門螺旋桿菌的假設性 parS 序列，皆位在複製起點附近 (origin-proximal) 20-30% 的範圍之內，與其他物種相似。此一結果以及本實驗室先前針對 Soj 與 Spo0J 所作的功能性分析提供了 parABS 系統存在於胃幽門螺旋桿菌中的有力證據。

Helicobacter pylori is a Gram-negative, microaerophilic human gastric pathogen which infects the gastric mucosa, causes gastritis and peptic ulcer disease, and is also an important risk factor for development of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. There was a lack of information on the progression to diseases by H. pylori, which survive at the acidic pH of the gastric environment, a site that few other microbes can colonize. The requirement of a rather stringent culture condition as compared to other bacteria, and the resistance to transformation in many strains, resulted in the lack of adequate systems for genetic manipulation. With the resolution of the complete genome sequence of H. pylori, studies on this etiological agent can be more conveniently implemented through the construction of recombinant proteins.
The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the production of spermidine after incubating the enzyme with putrescine and dcSAM. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695. The lack of a complete gatekeeping loop region in H. pylori spermidine synthase was suspected to be responsible for its instability in activity. Mutagenesis on conserved residues in the gatekeeping loop region of E. coli spermidine synthase (EcSDPS) was thus performed to investigate its importance to enzyme catalysis.
The inorganic pyrophosphatase of H. pylori (HpPPase) was encoded by the HP0620 (ppa) gene and was a family I PPase. It was a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of PPi by HpPPase relied on the presence of magnesium and followed Michaelis-Menten kinetics, with kcat being 344 s-1 and Km being 83 μM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substituting Cys15 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% of activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C15S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y139F HpPPase and those of E. coli. Although Cys15 was not located at the subunit interface of the hexameric HpPPase, several evidences suggested that the C15S substitution destabilized HpPPase through impairing trimer-trimer interactions. These results provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure.
The Soj and Spo0J proteins, together with one or more parS sequences, constitute the parABS system, which is crucial to chromosome segregation and the progression of cell cycle in many bacteria. A putative oriC region was located at 1,609,162 bp of the circular chromosome of H. pylori 26695. Genes coding for Soj (HP1139) and a plasmid replication-partition related protein containing a Spo0J or ParB conserved domain (HP1138), together with two putative parS sites identified by blasting the parS sequence of Bacillus subtilis against the genome of H. pylori 26695, were found to be located within the origin-proximal 20-30% of the chromosome, similar to several other species. These analyses, together with previous functional studies of recombinant Soj and Spo0J proteins, led to the first implication of the existence of a functional parABS system in H. pylori.

摘要 i
Abstract iii
Abbreviation List v
誌謝 vi
Table of Contents vii

Chapter 1 Introduction 1
1-1 Helicobacter pylori 1
1-2 Complete Genome Sequences of H. pylori 1
1-3 Physiologically Significant Proteins of H. pylori 2

Chapter 2 Spermidine Synthase 3
2-1 Introduction 3
2-1-1 Polyamine Biosynthesis 3
2-1-2 Importance of Spermidine Synthase 4
2-1-3 H. pylori Spermidine Synthase 5
2-2 Materials and Methods 7
2-2-1 Cloning, Expression, and Purification 7
2-2-1-1 Materials 7
2-2-1-2 Preparation of the HP0832 Insert 7
2-2-1-3 Preparation of the pQE30 Vector 9
2-2-1-4 Preparation of the pQE30-HP0832 Plasmid 10
2-2-1-5 Transformation of E. coli Strain SG13009 10
2-2-1-6 Analysis of Transformants 11
2-2-1-7 HP0832 Protein Expression and Purification 12
2-2-2 Antibody Production 13
2-2-3 H. pylori Strain and Growth Conditions 13
2-2-4 Immunoprecipitation 13
2-2-4-1 Preparation of the Media 13
2-2-4-2 Immunoaffinity Chromatography 14
2-2-5 Western Blot Analysis 14
2-2-6 Spermidine Synthase Assay 15
2-2-6-1 Materials 15
2-2-6-2 HPLC Analysis 15
2-2-6-3 TLC Analysis 16
2-2-7 Carboxynorspermidine Synthase Assay 16
2-2-8 Site-Directed Mutagenesis of EcSPDS 17
2-3 Results 18
2-3-1 H. pylori Spermidine Synthase 18
2-3-1-1 Cloning and Expression 18
2-3-1-2 Enzyme Purification and Characterization 18
2-3-1-3 Spermidine Synthase Assay 19
2-3-1-4 Intracellular Polyamine Content of H. pylori 19
2-3-1-5 Detection of Endogenous Spermidine Synthase in H. pylori 20
2-3-2 E. coli Spermidine Synthase 21
2-3-2-1 Characterization of EcSPDS 21
2-3-2-2 Site-Directed Mutagenesis of the Gatekeeping Loop Region 22
2-4 Discussion 23
2-4-1 Characterization of H. pylori Spermidine Synthase 23
2-4-2 Synthesis of dcSAM 24
2-4-3 Analysis of Polyamines 25
2-4-4 Polyamine Biosynthesis in H. pylori 26

Chapter 3 Inorganic Pyrophosphatase 28
3-1 Introduction 28
3-1-1 Family I and Family II Inorganic Pyrophosphatases 28
3-1-2 Activation of Inorganic Pyrophosphatase 28
3-1-3 Cysteine Residues and Thermostability 29
3-1-4 H. pylori Inorganic Pyrophosphatase 30
3-2 Materials and Methods 31
3-2-1 Enzyme Expression and Purification 31
3-2-2 Site-Directed Mutagenesis 31
3-2-3 Inorganic Pyrophosphatase Assay 31
3-2-4 Circular Dichroism Spectroscopy 32
3-2-5 Sedimentation 32
3-3 Results 33
3-3-1 Characterization of HpPPase 33
3-3-2 Activation of HpPPase 33
3-3-3 Effect of C15S Replacement on Enzyme Activity and Reductant Activation 34
3-3-4 Effect of C15S Replacement on Thermostability and Quaternary Structure 35
3-3-5 Effect of pH 37
3-3-6 Effect of Replacement on Conformation 37
3-4 Discussion 38

Chapter 4 Chromosome Segregation System 40
4-1 Introduction 40
4-1-1 parABS Chromosome Segregation System 40
4-1-2 parABS System in H. pylori 41
4-2 Results and Discussion 43
4-2-1 Nucleotide Sequence Analysis 43
4-2-2 Identification of oriC in H. pylori Strain 26695 43
4-2-3 Localization of parABS Relative to oriC 44

References 46
Figures 61
Tables 90
Publication List 98

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