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研究生:蔡東益
研究生(外文):Tung-Yi Tsai
論文名稱:Xanthomonascampestris之PqqD在pyrroloquinolinequinone生合成操縱組中採取新的馬鞍狀折疊且可能為PQQ的載體
論文名稱(外文):Xanthomonas campestris PqqD in the pyrroloquinoline quinone biosynthesis operon adopts a novel saddle-like fold that possibly serves as a PQQ carrier
指導教授:周三和
指導教授(外文):Shan-Ho Chou
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:61
中文關鍵詞:PqqD晶體結構十字花科黑腐病菌咯奎新的折疊馬鞍狀
外文關鍵詞:PqqD crystal structureXanthomonas campestrisPQQnovel foldsaddle-like
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Pyrroloquinoline quinone (PQQ)是一個新的對熱穩定,水溶性的氧化還原輔因子,存在於多種細菌的酒精去氫酶和醛糖去氫酶裡。它屬於pyridine nucleotide-(如NAD)和flavin-dependent(如FAD)輔因子之後的第三類ortho-quinone輔因子。PQQ的發現,也引起人們相當大的興趣,因為其同時在動物和植物中可當作生長促進因子。雖然許多利用PQQ當作輔因子的酵素其結構與功能已知,但對於PQQ的生合成路徑了解甚少。在克雷伯氏肺炎桿菌(Klebsiella pneumoniae)中,pqqABCDEF 操縱組的六個基因已被發現對於PQQ的生合成是必要的,PQQ的生合成被發現有著許多獨特的步驟。研究這些基因的結構對於了解生合成的機制是必要的。然而,至今,只有PqqB和PqqC的結構被解析出來。我們使用X-ray晶體繞射分析法解析出植物病原菌Xanthomonas campestris的PqqD晶體結構,其解析度達到1.66 Å。我們亦發現XcPqqD採取一新的二聚體馬鞍狀折疊,與先前研究所預測的結構明顯不同。此外,在結構中亦發現一磷酸根座落在帶有高度正電的中央通道。將PqqD與PQQ進行docking模擬研究後,發現PQQ可座落在相似於磷酸根結合部位,顯示PqqD可能與PQQ結合並且扮演PQQ載體的角色。
Pyrroloquinoline quinone (4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]quinoline- 2,7,9-tricarboxylic acid, PQQ) is a novel heat-stable, water soluble redox cofactor present in a variety of bacterial alcohol and aldose dehydrogenases. It belongs to the third family of ortho-quinone cofactor following the well-known pyridine nucleotide- (such as NAD) and flavin-dependent (such as FAD) cofactors. Discovery of PQQ has elicited considerable interests due to its role as a growth-promoting factor both in animals and plants. Although the structures and functions of many enzymes using PQQ as a cofactor has been elucidated, relatively little is known about the pathway leading to the biosynthesis of this intriguing cofactor. Recently six genes belonging to the pqqABCDEF operon were found to be essential for the biosynthesis of PQQ, and the biosynthesis of PQQ were found to involve many unique synthesis steps. Structural studies of these genes are necessary to elucidate the synthesis mechanism. However, until now, only the structures of PqqB and PqqC have been determined.We now report the tertiary structure of PqqD from a plant pathogen Xanthomonas campestris determined to a resolution of 1.66 Å using X-ay crystallography. The crystal structure of XcPqqD was found to adopt a dimer of novel saddle-like fold that reveal considerable differences to the structure predicted de novo and carried out in the absence of suitable template. In addition, a phosphate ligand was found to be situated very well in the highly positively-charged central channel. Docking study of PqqD with PQQ also located PQQ at the similar phosphate binding site, indicating that PqqD can possibly bind PQQ and serve as a PQQ carrier.
中文摘要 i
Abstract ii
目次 iii
圖表目次 vi
縮寫檢索表 viii
第一章 前言 1
(一)Pyrroloquinoline quinone(PQQ)生合成路徑介紹 1
(二)Xanthomonas campestris 之結構基因體計畫介紹 2
第二章 材料與方法 5
(一)基因轉殖 5
1. 染色體DNA之抽取 5
2. 引子的設計 5
3. 聚合酶連鎖反應 5
4. 定點突變 6
5. 膠體電泳 8
6. DNA之純化 9
7. 質體DNA之抽取 9
8. Ligation-Independent cloning(LIC) 9
9. E. coli 勝任細胞之製備 11
10. 轉殖作用 12
11. PCR check與DNA定序 12
(二)蛋白質之大量表現與純化 13
1. SDS-PAGE的製備 13
2. 蛋白質之大量表現 14
3. 蛋白質之初步純化 16
4. 以陰離子交換層析法純化蛋白 18
5. 蛋白質濃度測定 19
6. 分析型超高速離心實驗 19
(三)利用X-ray晶體繞射技術解析蛋白質之結構 20
1. 蛋白質結晶實驗 20
2. 結晶條件篩選 21
3. 手動微調和大量結晶 21
4. 晶體的保護 22
5. X-ray晶體繞射實驗 22
6. 決定相位角 23
7. 結構的構築與修正 25
(四)蛋白質與小分子嵌合的研究分析 25
第三章 結果與討論 26
(一)XcPqqD基因轉殖、蛋白質表現及純化 26
(二)XcPqqD蛋白質之結晶與X-ray晶體繞射實驗 27
(三)整體結構 28
(四)二聚體界面間的交互作用 28
(五)XcPqqD可能的功能 29
(六)XcPqqD蛋白質的折疊方式 30
第四章 結論 31
第五章 參考文獻 32
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