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研究生:溫皓宇
研究生(外文):HaoYu Wen
論文名稱:利用飽和定點突變針對氧化鯊烯環化酵素內假設活性區域胺基酸進行結構─反應之研究
論文名稱(外文):Studies of structure-reactivity relationships on pautive active site cavity residues of oxidosqualenen-lanosterol cyclase by site-saturated mutagenesis
指導教授:吳東昆
指導教授(外文):Tung-Kung Wu
學位類別:碩士
校院名稱:國立交通大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:英文
論文頁數:74
中文關鍵詞:氧化鯊烯環化酵素飽和定點突變
外文關鍵詞:oxidosqualene cyclasesite saturated mutagensis
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氧化鯊烯環化酵素(oxidosqualene-lanosterol cyclase, ERG7)在動物、真菌及高等植物體內催化非環狀的氧化鯊烯 (oxidosqualene)環化成為四環或五環的三菇帖類(Triterpnoids)。由氧化鯊烯環化酵素所一步催化而產生的一連串環化/重排反應,在這過半個世紀以來都一直令有機生物學家著迷。在本研究課題中,我們已經成功將啤酒酵母菌(Saccharomyces cerevisiae)中的Tyr510和Phe699等位置適合在羊毛脂醇生成中扮演穩定碳陽離子的轉移和成環反應之芳香族胺基酸,做飽和定點突變並進行反應與結構關係相關研究。在ERG7Y510X的飽和定點突變株內產生包含achilleol A、camelliol C、 (13αH)-isomalabarica-14(26)E,17,21-trien-3β-ol、lanosterol和parkeol等產物。這些結果暗示Tyr510可能扮演了穩定在環化/重排反應串中所產生的單環C-10碳陽離子、反馬可尼可夫C-14碳陽離子(anti-Markevoikov C-14 cation)以及羊毛脂醇碳陽離子中間物(lanosteryl cation)的重要角色。

另外在ERG7F699X的飽和定點突變株實驗中,除了Thr、Met、Pro、Leu、Ile、His等胺基酸取代基外,絕大部分的取代基都喪失了完整的環化酵素功能,這結果暗示該殘基位置對氧化鯊烯環化酵素的重要性。近一步地,我們首次在ERG7突變株發現三種新產物包含 prososta-13(17),24-dien-3β-ol、 prososta-17(20),24-dien-3β-ol和malabarica-14E,17,21-trien-3β-ol等。除此之外還包括 (13αH)-isomalabarica-14Z,17,21-trien-3β-ol與(13αH)-isomalabarica-14E,17,21-trien-3β-ol這些在椅式─船式(C-B)或椅式─椅式 6-6-5三環碳陽離子或羊毛脂醇碳陽離子環化重排時提前終止所產生的中間產物。整合以上結果證明了Phe699在氧化鯊烯環化酵素內不僅扮演了穩定反馬可尼可夫C-14碳陽離子以及羊毛脂醇C-17碳陽離子中間物外更擁有改變氧化鯊烯環化酵素內活性區結構及其之後的反應的可塑性。
Oxidosqualene-lanosterol cyclases (ERG7) catalyze the conversion of acyclic oxidoaqualene into tetracyclic or pentacyclic triterpenoids in the animals, fungi, and high plants. A serial cyclization/rearrangement cascades catalyzed in one-step reaction by oxidosqualene-lanosterol cyclase have fascinated the bioorganic researcher over a half century. We have successfully performed the site-saturated mutagenesis on the Tyr510 and Phe699 residues of Saccharomyces cerevisiae ERG7, corresponding to the appropriate orientations to stabilize the carbocation intermediates in the lanosterol formation. The site-saturated ERG7Y510X mutants produced achilleol A, camelliol C, (13αH)-isomalabarica-14(26)E,17,21-trien-3β-ol, lanosterol and parkeol. The results suggested that the position of Tyr510 residue may play a crucial role in stabilizing the monocyclic C-10 cation anti-Markovnikov C-14 cation intermediate and lanosteryl cation that were generated in the cyclization/rearrangement cascades.

In the cases of the site-saturated ERG7F699X mutants, most of the substituted residues failed to complement the cyclase activity, except the Thr, Met, Lys, Asn, and His, indicating the importance of the position for the catalytic function of ERG7. Further, three novel intermediates, protosta-13(17),24-dien-3β-ol, protosta-17(20),24-dien-3β-ol, and malabarica-14E,17E,21-trien-3β-ol, were isolated, for the first time, from the ERG7F699M mutant. These products, in addition to (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol and (13αH)-isomalabarica-14E,17E,21-trien-3β-ol, corresponding to truncation of the cyclization/rearrangement cascade at the chair-boat (C-B) or chair-chair (C-C) 6-6-5 tricyclic cation and/or lanosteryl C-17 cation. Taken together, these results demonstrate the functional role of the ERG7 Phe699 residue in stabilizing Markovnikov tricyclic C-14 and lanosteryl C-17 cations as well as the plasticity of the ERG7 mutant enzyme in changing the active site structure and subsequent reaction cascade.
Contents

Abstract (Chinese)…………………………….…………………………………...…Ⅰ
Abstract (English)…………………………………….….……………………....……Ⅲ
Acknowledgement …………………………………………………………………….Ⅴ
Abbreviations ………………………………………………………………………....Ⅵ
Table of Content ……………………………………………..………..........................Ⅶ
List of Figures….……………………………………………………………………...Ⅹ
List of Tables .………………………………...………….……….…….………..……XI
List of Schemes….……………………………………...………………….………....XII

Chapter 1 Introduction and Background
1.1 Sterol……………………………………………….…………...…...…...................1
1.2 Triterpene cyclases………...………………………......…...…………………...…4
1.3 Oxidosqualene-lanosterol cyclase…………………...…………………………....5
1.3-1 Mechanism of cyclization………………...…..……….…….. …………….5
1.3-2 The studies on site-directed mutagenesis………………………………...10
1.4 Cycloartenol synthase……...……………………………………………………...14
1.4-1 Mechanism of cyclization……………………………….…………………14
1.4-2 The studies on site-directed mutagenesis………………………………...15
1.5 Squalene-hopene synthase…...……………………....……..…………………….19
1.5-1 The studies on site-directed mutagenesis………………………………...21
1.6 Sequence alignment and homology modeling…………………………………...23
1.7 Research Goal..……………………………………………………………………27

Chapter 2 Materials and Methods
2.1 Chemicals and reagents……………………………………………………….….29
2.2 Bacterial, yeast strains, plasmids………………………………………………...30
2.3 Kits….…………….…………………………..……………………………....……31
2.4 Equipments………………………………………………………………………..31
2.5 Solution…………………………………………………………………….………32
2.6 Construction of mutants……………………………………………………….…35
2.6-1 Site-directed mutagenesis of ERG7Y510X and ERG7F699X………..….......35
2.6-2 Transformation and enzyme mapping.......................................................36
2.6-3 Sequence analysis of ERG7Y510X and ERG7F699X mutant gene….……...36
2.7 Transformation and live/die selections ERG7 mutants………………………...37
2.7-1 Preparation of TKW14C2 as competent cell………………………….…37
2.7-2 Transformation of mutated plasmid into TKW14C2…..…………….…38
2.7-3 Ergosterol supplement…………………………………………………….38
2.8 Extracting lipids and characterizing mutant product profiles………………...39
2.8-1 Cell culture and extraction………………………………………………..39
2.8-2 GC and GC/MS conditions………………………………………………..39
2.9 Isolation of mutant products by using acetylation modification………...……..40
2.9-1 Acetylation modification and the alkaline hydrolysis reaction…………40
2.9-2 Argentic colum chromatography…………………………………………40
2.9-3 Deacetylation reaction of the modified products………………………..40
2.10 Molecular modeling……………………………………………………….……..41

Chapter 3 Results
Part A: The functional analysis of tyrosine 510 within S.cerevsisiae ERG7
3A.1 Site-saturated mutagenesis of Tyr510……………………………….……42
3A.2. The result of homology modeling of S.cerevisiae ERG7.……………….47
3A.3. The double mutagenesis of ERG7H234W/Y510V and ERG7H234W/Y510W.......51
Part B: The functional analysis of phenylanaline 699 within S.cerevsisiae ERG7
3B Site-saturated mutagenesis of Phe699………………………………...……53

Chapter 4 Discussion and Conclusion…………………………………….…67
Chapter 5 Future Work…………………………………………………………71
Reference……………………………………………………………………………72
Appendix………………………………………………………………………….…75
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