臺灣博碩士論文加值系統

萜類合成酶(terpene synthases)的蛋白質結構與其功能性間之關聯性，長期以來一直是相關酵素改質工程所面臨的最大挑戰。本研究首先藉由設計退化性引子(degenerate primer)、聚合酶連鎖反應(polymerase chain reaction, PCR)與cDNA末端快速增幅(rapid amplification of cDNA ends, RACE)等方式，順利地自臺灣杉(Taiwania cryptomerioides)葉部選殖到單萜合成酶基因Tc-αpin/teo與Tc-teo；再透過比較兩者胺基酸序列與產物上的差異性，輔以同源模擬(homology modeling)及定點突變(site-directed mutagenesis)等技術，成功地在Tc-αPIN/TEO蛋白質結構中，界定出三個對萜類(terpenes)二次環化機制(secondary cyclization mechanism)有決定性影響力的塑性殘基(plasticity residues)，分別為Y327、Y429與Y575。將這些tyrosine突變成phenylalanine的試驗結果顯示，其胺基酸支鏈(side chain)上之hydroxyl groups，可能藉由協同作用(synergistic effect)共同幫助碳陽離子(carbocation)進行二次環化。這些發現，或許能使我們對於複雜的萜類生合成步驟有更深入的瞭解，並作為未來蛋白質工程(protein engineering)試驗設計之參考。

The ambiguous protein structure-function correlation of terpene synthases has been a challenge for protein engineers. Thus, we intended to decipher the determinants associated with the product specificity of terpene synthases. Using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods, we successfully cloned two monoterpene synthase genes, Tc-αpin/teo and Tc-teo, from Taiwania cryptomerioides. The enzymes encoded by these genes shared 97% similarity of amino acid sequences, but had different terpene product profiles. We adopted homology modeling to compare the structural differences between Tc-αPIN/TEO and Tc-TEO, and successfully identified three plasticity residues involved in secondary cyclization around the active site of Tc-αPIN/TEO, namely Y327, Y429 and Y575. In the site-directed mutagenesis experiments, converting these phenolic residues into phenylalanines resulted in a dramatic decrease in the synthesis of α-pinene, indicating that the hydroxyl group of tyrosine may play an important role in the synthesis of bicyclic terpene products. This finding may assist our understanding of the complex cyclization processes and protein engineering design in the future.

口試委員會審定書 #
誌謝 i
中文摘要 v
Abstract vi
目錄 vii
圖目錄 x
表目錄 xiii
一、 前言 1
1.1 萜類(Terpenes)與萜類合成酶(Terpene synthases, TPS) 1
1.2 萜類之應用 1
1.3 臺灣杉與萜類合成酶基因家族之演化地位 2
1.4 本研究之目的 3
二、 文獻探討 4
2.1 裸子植物萜類合成酶之分群 4
2.2 裸子植物單萜合成酶基因之內隱子分布情形 5
2.3 單萜生合成路徑 6
2.4 單萜生合成機制 7
2.5 萜類合成酶的蛋白質結構 11
2.5.1 引發催化反應之重要功能區域(Catalysis-initiating motifs) 12
2.5.2 具穩定碳陽離子能力之殘基(Carbocation-stabilizing residues) 15
2.5.3 演化保守的Double-arginine motif (RRX8W)與RXR motif 17
2.5.4 鉀離子結合區域(Potassium-binding region) 19
2.6 萜類合成酶蛋白質結構─功能模式 21
三、 試驗材料與方法 24
3.1 植物試材 24
3.2 臺灣杉單萜合成酶基因之選殖 24
3.3 單萜合成酶基因內隱子之取得 28
3.4 裸子植物萜類合成酶相似度樹狀圖(Dendrogram)分析 28
3.5 葉綠體運輸胜肽及蛋白質分子量(Mw)、等電點(pI)之預測 30
3.6 蛋白質表現質體之構築(Construction) 31
3.7 西方墨點雜合分析(Western blotting analysis) 31
3.8 重組蛋白質(Recombinant protein)誘導大量表現 33
3.9 目標蛋白質之純化 33
3.10 目標蛋白之萜類產物分析(GC-MS) 34
3.11 蛋白質結構模擬 34
3.12 定點突變試驗(Site-directed mutagenesis) 35
四、 結果 39
4.1 臺灣杉單萜合成酶基因Tc-αpin/teo與Tc-teo 39
4.2 Tc-αpin/teo與Tc-teo內隱子分布情形 41
4.3 裸子植物萜類合成酶相似度樹狀圖分析 43
4.4 Tc-αPIN/TEO與Tc-TEO蛋白質純化與活性反應分析 45
4.5 Tc-αPIN/TEO與Tc-TEO模擬蛋白質結構 46
4.6 定點突變試驗 48
五、 討論 57
5.1 Tc-αpin/teo與Tc-teo之內隱子分布情形 57
5.2 裸子植物萜類合成酶相似度樹狀圖分析 58
5.3 Tc-αPIN/TEO與Tc-TEO之蛋白質特性 61
5.4 萜類合成酶蛋白質結構與二次環化機制之相關性 68
5.5 Terpinolene與α-pinene可能的生合成機制 72
5.6 Terpinolene與α-pinene的生物活性及相關應用 74
六、 結論 76
七、 參考文獻 77

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