尿嘧啶核苷酸激酶(UMP kinase)在細菌中是個生存基本的酵素，它參與有關細菌內部的嘧啶生合成路徑(De novo synthesis pathway of pyrimidine nucleotide)。尿嘧啶核苷酸激酶是的作用是將三磷酸腺苷酸(ATP)上的磷酸基團轉移到尿嘧啶核苷酸(UMP)上，最終形成雙磷酸尿嘧啶核苷酸(UDP)及雙磷酸腺苷酸(ADP)。在胃幽門螺旋桿菌裡，尿嘧啶核苷酸激酶是由pyrH 基因所轉錄出來，並含有240個殘基。在本研究當中，pyrH 的產物（HpUMPK）被予以大腸桿菌表現系統所表現，並純化得到單一分子量為25.8kDa的HpUMPK，並證實其也是以六聚體的形式存在於水溶液中。我們以PEG MME 550以主沉澱劑結晶出HpUMPK-UDP複合物的晶體，並得到解析度2.45Å的X光繞射數據。此晶體為C2對稱性排列，其單位晶格參數為a = 147.5 Å, b = 127.6 Å, c = 93.5 Å, β = 90.9°, 不對稱單元中具有六個分子。我們利用序列一致性約49%的大腸桿菌尿嘧啶核苷酸激酶，作為分子置換法(molecular replacement, MR)的搜索模型，以解決相位角的問題。在晶體結構中，HpUMPK是以六聚體的形式存在，並且此六聚體是以三個雙聚體的方式疊合而成。整個蛋白桔構是由九個α螺旋及七個β摺板所組成，而UDP則是位在中央的β折疊上方。同時，我們也在此針對HpUMPK與其他原核物種的尿嘧啶核苷酸激酶以及真核生物體中的同功酶嘧啶核苷酸激酶作比較。

Contents
中文摘要 i
Abstract ii
Contents iii

Chapter 1 Introduction
1.1 Helicobacter 1
1.2 UMP kinase 2
i. Background for NMP kinases 2
ii. The characteristics of UMP kinase 2
iii. The UMP kinase in E. coli 3
iv. The UMP kinse of Helicobacter pylori 4

Chapter 2 Materials and Methods
2.1 Protein expression 6
2.2 Purification and Crystallization 6
2.3 Data collection and Process 7
2.4 Self-rotation functions 7
2.5 Structure determination and Refinement 8

Chapter 3 Results
3.1 Multiple sequence alignment 9
3.2 Protein preparation, purification, and characterization 9
3.3 Crystallization 10
3.4 X-ray diffraction data collection and space group determination 10
3.5 Phase determination of HpUMPK 11
3.6 Model building 11
3.7 Structural refinement 12
3.8 Overall structure of HpUMPK-UDP complex 12
3.9 Oligomeric structure of HpUMPK-UDP complex 12
Chapter 4 Discussion
4.1 The overall structure of HpUMPK 14 4.2 The UDP free form and the UDP bound form 15
4.3 The ATP and allosteric binding sites 17
4.4 Comparison between HpUMPK, prokaryotic UMPK and eukaryotic UMP/CMP kinases 18

Figures 20
Tables 43
References 51

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