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研究生:陳志豪
研究生(外文):Chen, Chih-Hao
論文名稱:幽門螺旋桿菌磷酸泛酸醯基乙胺腺苷轉移酶結構與功能之研究
論文名稱(外文):Structural and Functional Studies of Phosphopantetheine aenylyltransferase from Helicobacter pylori
指導教授:殷献生
指導教授(外文):Yin, Hsien-Sheng
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:2009
畢業學年度:97
語文別:英文
論文頁數:47
中文關鍵詞:幽門螺旋桿菌輔酶A磷酸泛酸醯基乙胺腺苷轉移酶
外文關鍵詞:Helicobacter pyloriCoenzyme APhosphopantetheine adenylyltransferase
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世界上估計將近有一半的人口都被幽門螺旋桿菌寄生著,它生活在人體的胃部及十二指腸。感染幽門螺旋桿菌會引發胃及十二指腸慢性發炎,長期的發炎會導致潰瘍的產生。由於抗生素的頻繁使用,對於抑制幽門螺旋桿菌已經造成效力減弱的結果。因此在抑制的機制上轉為研究專門對抗其生理代謝路線的方法。輔酶A是在所有生物的代謝上都會到用的醯基攜帶者,它牽涉到接近5%的生理代謝反應。在前人的研究發現抑制輔酶A的生合成可以有效達到抑制細菌生長的效果。輔酶A的生合成包含著五個步驟,其中倒數第二個步驟是一個速率決定步驟。這個步驟由酵素磷酸泛酸醯基乙胺腺苷轉移酶 (phosphopantetheine adenylyltransferas) (PPAT) 催化,並且會被下游合成的輔酶A來抑制其活性。PPAT在細菌裡有著很高的序列相似性,但是與人類相比卻沒有顯著的相似序列,因此發展對抗此酵素的藥物對於抑制幽門螺旋桿菌有很大的幫助。在研究此酵素的結構上使用了X-光結晶學的方法,得到了幽門螺旋桿菌PPAT的晶體,並且解出其結構。這個結構是一個open form 的構形,這是在其他物種的PPAT上沒見過的。本文使用圓二色光譜來探討PPAT的二級結構的穩定性,另外也使用了�皕驕w定熱卡計來分析其酵素催化能力。
Coenzyme A (CoA) is the principle acyl carrier in all living cells, and is required for many metabolic reactions, including citric acid cycle and fatty acid metabolism. Phosphopantetheine adenylyltransferase (PPAT; EC: 2.7.7.3) is an essential enzyme that catalyzes the penultimate step of CoA biosynthesis by transferring an adenylyl group from ATP to 4’-phosphopantetheine (Ppant), yielding 3’-dephospho-CoA (dPCoA) and pyrophosphate. PPAT catalyzes the second rate-limiting step in CoA synthetic pathway and is regulated by feedback inhibition by downstream end product, CoA. The bacteria PPAT sequences show high degree of homology but are dissimilar from mammalian, therefore, this enzyme might be a good antibacterial target. PPAT from Helicobacter pylori (H. pylori) which infects about 50% world population and cause gastric and duodenal ulcers was expressed and crystallized in space group I222. The crystal structure of H. pylori PPAT has been determined by molecular replacement method at 1.75 Å resolution. The structure shows an open form of H. pylori PPAT that have not been observed in other PPATs. Since previous reports have shown that PPAT from Escherichia coli exhibites pH-dependent binding affinity towards substrates, the PPAT structural stability and activity was studied by circular dichroism and isothermal titration calorimetry.
Chapter 1.................................................................................................................................. 1
Introduction.......................................................................................................................... 1
1.1 Helicobacter pylori....................................................................................................... 1
1.2 Coenzyme A................................................................................................................. 2
1.3 Phosphopantetheine adenylyltransferase .................................................................. 3
Chapter 2.................................................................................................................................. 6
Materials and methods ......................................................................................................... 6
2.1 Cloning of the coaD gene from H. pylori ..................................................................... 6
2.2 Over-expression of the coaD gene from H. pylori ....................................................... 6
2.3 Purification of recombinant H. pylori PPAT................................................................. 7
2.4 Absorption spectroscopy............................................................................................. 7
2.5 Fluorescence spectroscopy ......................................................................................... 8
2.6 Isothermal titration calorimeter experiment .............................................................. 9
2.7 Analytical gel-filtration chromatography .................................................................... 9
2.8 Circular Dichroism spectrum experiment.................................................................... 9
2.9 H. pylori PPAT crystallization..................................................................................... 10
2.10 X-ray diffraction data collection.............................................................................. 11
2.11 Molecular replacement ........................................................................................... 11
2.12 Crystallographic refinement.................................................................................... 12
Chapter 3................................................................................................................................ 13
Results ................................................................................................................................ 13
3.1 Cloning of the coaD gene from H. pylori. .................................................................. 13
3.2 Over-expression and purification of H. pylori PPAT .................................................. 13
3.3 Characterization of H. pylori PPAT and N76Y H. pylori PPAT .................................... 14
3.4 Determination of bound CoA .................................................................................... 15
3.5 Determination of substrate ATP binding................................................................... 15
3.6 Isothermal titration calorimeter experiment ............................................................ 16
3.7 Secondary structure and thermal stability analysis .................................................. 16
3.8 The crystal structure of N76Y H. pylori PPAT ............................................................ 17
Chapter 4................................................................................................................................ 19
Discussion........................................................................................................................... 19
4.1 H. pylori PPAT active site ........................................................................................... 19
4.2 The broken electron density map ............................................................................. 20
4.3 Secondary structure change...................................................................................... 20
Figures and figure legends ...................................................................................................... 22
Tables...................................................................................................................................... 42
References.............................................................................................................................. 44
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