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研究生:劉媛婷
論文名稱:利用突變策略針對氧化鯊烯環化酵素進行其結構與功能及其產物專一性/多樣性之研究
論文名稱(外文):Mutagenesis Approach to Investigate the Structure-Function Relationships and Product Specificity/Diversity of Oxidosqualene Cyclase
指導教授:吳東昆
指導教授(外文):Wu, Tung-Kung
學位類別:博士
校院名稱:國立交通大學
系所名稱:生物科技學系
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:2010
畢業學年度:98
語文別:英文
論文頁數:128
中文關鍵詞:氧化鯊烯環化酵素突變效應
外文關鍵詞:oxidosqualene cyclaseMutagenesis
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突變策略被視為是一種強而有力的工具,其廣泛地應用於針對蛋白質的結構、功能、或反應機制的研究上。因此,在本論文中我們透過某些功能性已知的環化酵素,對於其胺基酸序列和蛋白質結構間的關係進行比較,並利用丙胺酸掃描式突變(alanine-scanning) 或定點/飽和突變的策略 (site-directed/saturated mutagenesis) 並配合基因互補和產物收集/鑑定的方法,針對啤酒酵母菌中的氧化鯊烯環化酵素(Saccharomyces cerevisiae ERG7) 其活性區域內多個重要的胺基酸位置,它們在環化酵素所負責的複雜環化與重組反應機制中所扮演的角色加以確定。一開始的工作是針對活性區域內的組胺酸-234號位置。當此位置被置換成其它不同性質的胺基酸時,我們透過所獲得的多樣性產物,包括單環、三環、和不同去質子化的四環產物進一步地闡明此胺基酸參予在環化酵素所催化之反應中的角色。此外,利用相似的突變策略對於其它重要的胺基酸位置,我們可以透過來自各突變株所收集到的不完全環化產物或是不同重組階段的替代產物加以解釋各個胺基酸所賦予在各環化重組過程中的重要性。同時,為了要更進一步的細看最後的去質子步驟,對於決定環化酵素其產物特異性的關係,我們建立多個連續定點突變株。其中一個突變酵素上第384號位置的蘇胺酸我們將之換成酪胺酸,而谷氨醯胺450位置將其換成組胺酸,最後我們將纈胺酸454位置換成異亮胺酸 (也就是我們同時突變這三個胺基酸位置)。由實驗的結果發現,此突變株劇烈地改變它的特異性,從原本的羊毛硬脂醇合成酵素變成一個極度精密的帕克醇合成酵素,另外我們亦發現其產物中伴隨著兩個骨架已被下游連續酵素所修飾的產物。這個現象不僅反映當有不尋常量的三萜類化合物累積在酵母菌體時,其代謝流向的改變情況,而且也說明了下游酵素對於受質的選擇性效應。除了上述的突變實驗外,我們也同步針對植物中的一個三萜類合成酵素,即 刍-香桂素合成酵素進行系列的研究。包括從碗豆 (Pisum sativum) 種子中進行分子選殖以獲得 刍-香桂素合成酵素的基因,並利用異質性的表現系統將其成功地在酵母菌中表現,其後我們利用電腦模擬的方式建立其同源性結構,並透過上述的定點突變實驗對於其重要的19個胺基酸位置,進行研究並分析突變效應後所產生的環化產物的樣式。雖然我們並沒有發現在各個突變株中有差異的非皂化脂質粗萃取物,但這一系列的實驗說明當 刍-香桂素合成酵素中的重要胺基酸被置換時,會顯著地影響此酵素之催化活性。因此,透過上述的實驗結果我們可以對於不同的氧化鯊烯環化酵素其在環化/重組反應機制上提供更多的了解。
The mutagenesis is regarded as a powerful tool for investigating the structure, function, and reaction-mechanism relationships of proteins. Sequence alignment with other known cyclases and various forms of mutagenesis were used to identify and study catalytically important residues in Saccharomyces cerevisiae ERG7. Using mutagenic techniques coupled with genetic complementation and product characterization, it became possible to further characterize the cyclization and rearrangement mechanism of 2,3-oxidosqulene. Several mutations of the His-234 residue in ERG7 generated diverse product profiles with various monocyclic, tricyclic, and tetracyclic products while similar mutagenesis strategies on other catalytically important residues resulted in derailed cyclization and alternative deprotonation in other critical steps of the cyclization cascade. A series of site-directed mutations were also made to probe the crucial residues involved in the final deprotonation stage of the reaction for product specificity. For example, the ERG7T384Y/Q450H/V454I mutant changed its product specificity from the original lanosterol synthase into a parkeol synthase accompanied by two scaffold-modifying products that were generated speculatively by additional tailoring enzymes. The trend of metabolite flux changed when unusual levels of triterpene accumulated in the yeast cell and impacted the substrate selectivity of some downstream enzymes. A parallel experiment was conducted with a triterpene synthase, 刍-amyrin synthase from Pisum sativum using functional expression by yeast and site-directed mutagenesis on 19 residues. Examination of its product profile revealed no divergence of the non-saponifiable lipid patterns among any of the mutants, suggesting that the exchange of the important functional residue of 刍-amyrin synthase might influence its enzymatic activity dramatically. The aforementioned results, when combined, provides for a better understanding for the cyclization and rearrangement mechanism of various oxidisqualene cyclases.
Chapter 1. General introduction. 1
1.1 Overview of oxidosqualene cyclase 1
1.2 Historical hypothesis of the cyclization mechanism 5
1.2-1 Mechanistic and stereochemical insights relative to the 2,3-oxidosqualene
cyclization cascade 5
1.2-2 The theoretical models of cylase enzymes 11
1.3 Crystallization and structural characterization of cyclase 15
Chapter 2. Thesis organization 21
Chapter 3. Site-deirected mutagenesis of oxidosqualene cyclase to characterize the plasticity of the protein diversity of product 24
3.1 Research background and aim 24
3.2 Results and Discussion. 27
3.2-1 Alignments of multiple amino acid sequences for sterol cyclases and triterpene cyclases. 27
3.2-2 Nine site-directed mutants of ERG7 gene from S. cerevisiae 28
3.2-3 Principle of the plasmid shuffle method 31
3.2-4 Screening inactive ERG7 mutants via plasmid shuffle method 33
3.2-5 Lipid extraction, column chromatography, and product characterization of yeast transformants 34
3.2-6 Characterizatio of the mutant products in the novel gene disruption strain, TKW14c2 strain 37
3.2-7 Site-saturated mutagenesis of His-234 to investigate its importance for ERG7 activity 40
Chapter 4. Site-directed mutagenesis and product characterization to study the putative active-site residues from S. cerevisiae oxidosqualene cyclase 52
4.1 Research background and aim. 52
4.2 Results and Discussion. 53
4.2-1 Generation of a homology model for S. cerevisiae OSC and functional analysis of alanine-scanning mutants of putative active-site residues 53
4.2-2 Site-saturated mutagenesis and functional analysis of active-site residues from S. cerevisiae ERG7.. 55
4.2-3 Correlation between theoretical model results and experimential evidance to investigate cyclization/rearrangement mechanism of oxidosqualene cyclase. 59
4.3 Conclusion. 65
Chapter 5. Site-directed mutagenesis study on the deprotonation course of the oxidosqualene cyclization 66
5.1 Research background and aim 66
5.2 Results and Discussion. 69
5.2-1 Generation of site-directed mutants on the S. cerevisiae erg7 gene and functional analysis of site-directed mutations via plasmid shuffle method and product characterization 69
5.2-2 Homology modeling illustration of critical residues on enzyme function 73
5.2-3 Investigation of product modification by subsequent triterpene tailoring enzymes in S.cerevisiae by isolation and identifying unexpected downstream products. 74
Chapter 6. Homology modeling coupled with site-directed mutagenesis study on plant oxidosqualene 刍-amyrin synthase to investigate the relationship between substrate folding geometry and the resulting diverse products 82
6.1 Research background and aim. 82
6.2 Results and Discussion. 85
6.2-1 Putative 刍-amyrin synthase cDNA amplified from P. sativum. 85
6.2-2 Heterologous expression of 刍-smyrin synthase in yeast 87
6.2-3 Study of enzyme activity using homology modeling coupled with site-directed mutagenesis approach. 89
6.2-4 Functional analysis of artificial enzymes via plasmid shuffle method and the product characterization 93
6.3 Conclusion. 96
Chapter 7. Future perspective 98
Chapter 8. Experimental section 101
8.1 Material. 101
8.1-1Bacterial strains and molecular cloning/expression vectors. 101
8.1-2 Enzyme, chemicals, equipments, and reagents 101
8.2 general experimental procedure. 104
8.2-1 Construction of site-directed/saturated mutahenic plasmids. 104
8.2-2 Preparation of competent yeast cell (CBY 57 and TKW14c2 strain) 107
8.2-3 Cyclase activity assay by using plasmid shuffle method in CBY57 strain 107
8.2-4 Cyclase activity assay by using ergosterol compementation in TKW14c2 strain. 108
8.2-5 Lipid extraction, and column chromatography 109
8.2-6 Acetylationmodification and the alkine hydrolysis reaction. 109
8.2-7 GC and GC-MS column chromatography 110
8.2-8 Sequence alignment and molecular modeling 110
8.2-9 Molecular cloning of P. sativum 刍-amyrin synthase. 111
8.2-10 Subcloning of full-length of P. sativum 刍-amyrin synthase 112
8.2-11 Functional expression of P. sativum 刍-amyrin synthase 113
References. 114
Appendix. 119

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