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研究生:簡湘誼
研究生(外文):Shiang-Yi Chien
論文名稱:幽門螺旋桿菌亞精胺合成酶晶體結構與功能之研究
論文名稱(外文):Crystal Structure and Functional Studies of Spermidine Synthase from Helicobacter pylori
指導教授:孫玉珠
指導教授(外文):Yuh-Ju Sun
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:55
中文關鍵詞:幽門螺旋桿菌亞精胺合成酶晶體結構
外文關鍵詞:Helicobacter pylorispermidine synthaseCrystal Structure
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在生物體內普遍存在著各種多胺分子(polyamines),例如腐胺(putrescine)、亞精胺(spermidine)與精胺(spermine)等。這些多胺分子帶正電的特性能平衡細胞內巨分子的電荷,包括DNA、RNA以及蛋白質分子都深受其影響。因此,當細胞進行分裂或分化時,多胺分子的調節是重要的關鍵。多胺分子的合成過程主要由三個酵素來調控;亞精胺合成酶是其中之一。該酵素的作用即是將decarboxylated S-adenosylmethionine的丙胺基(aminopropyl group)轉移到腐胺上,形成亞精胺分子。幽門螺旋桿菌的亞精胺合成酶具有262個氨基酸,其分子量約為30.5 kDa。我們利用X光晶體繞射實驗來決定幽門螺旋桿菌亞精胺合成酶的立體結構,以進一步瞭解它的生物功能。我們純化並結晶出含重金屬硒(selenium)的甲硫酸胺衍生物晶體,並透過多波長異常色散法(multiwavelength anomalous dispersion, MAD)來解決相位問題。經過分子模型的建立與修正後,於解析度為2.0 Å的情況下得到的R與Rfree值分別為23.6%和29.5%。仔細分析幽門螺旋桿菌亞精胺合成酶的立體結構,我們發現該酵素的四級結構為二元體(dimer)。每個結構單元(monomer)分成兩個區域:C端的Rossmann-like折疊結構,以及N端6股摺板區。其中,較大的C端區域是受質(substrates)進行催化、反應的空間;N端則扮演維持二元體結構的角色。
Polyamines, such as putrescine, spermidine, and spermine, are polycationic mediators of cell proliferation and differentiation in most organisms. Spermidine synthase catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine in the biosynthesis of spermidine. Here we report the crystal structure of spermidine synthase from Helicobacter pylori, which specialized in the colonization of the human stomach. Spermidine synthase from H. pylori contains 262 amino acids with a molecular weight of 30.5 kDa. Its crystal structure has been determined to 2.5Å resolution by multiwavelength anomalous dispersion (MAD) from selenomethionine (Se-Met)-containing proteins and refined to a crystallographic R-factor of 23.6% and an Rfree value of 29.5% so far. The enzyme shows the architecture of a dimer, and each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal beta-stranded domain. The larger C-terminal is a catalytic site and the N-terminal plays a role in maintaining the dimer structure.
Chapter 1 Introduction.....1
1-1 Helicobacter pylori
1-2 The Genome of Helicobacter pylori Strain 26695
1-3 The Target Gene HP0832 in Helicobacter pylori Strain 26695
1-4 Polyamine Functions and Biosynthesis
1-5 Spermidine Synthase

Chapter 2 Materials and Methods.....6
2-1 Cloning
2-2 Protein Expression and Purification
2-3 Gel Filtration Chromatography
2-4 Analytical Ultracentrifugation
2-5 Crystallization
2-6 X-ray Data Collection
2-7 Self-Rotation Functions
2-8 Phasing
2-9 Model Building and Refinement

Chapter 3 Results.....12
3-1 Protein Characteristics
3-2 Crystallization
3-3 Data Collection and Space Group Determination
3-4 Non-Crystallography Symmetry
3-5 Structure Determination
3-6 Model Building and Refinement
3-7 Overall Structure of H. pylori PAPT

Chapter 4 Discussion.....20
4-1 Amino Acids Sequence Alignment
4-2 Comparison of the TmPAPT and H. pylori PAPT Structures
4-3 Possible Catalytic Mechanism

Appendix Figures and Tables.....23
References.....53
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