臺灣博碩士論文加值系統

蚜蟲是種媒介昆蟲，除了會吸取寄主植物的養份外，還會對非寄主植物傳播病毒病，造成農業經濟的衝擊。蚜蟲以剌吸式口器從植物韌皮部吸食汁液為生，然而韌皮部含氮養份不足，故在蚜蟲腹腔的懷菌細胞中，具有內共生菌Buchnera，以提供宿主蚜蟲必需胺基酸、cysteine等攝取不足及無法自行合成的營養需求。
PDT (prephenate dehydratase )是由 pheA 所編譯出來的酵素，催化prephenate進行脫水反應與脫羧反應，使之轉變成phenylpyruvate，再經由芳香族轉胺酶催化而合成 phenylalanine，最後會再由終產物phenylalanine回饋抑制PDT。
在結構上，PDT 可細分為位於N端的catalytic domain，是受質prephenate結合的位置；與位於C端的requlatory domain，是由ACT domain (Aspartokinase, Chorismate mutase, TyrA) 所構成，以dimer的狀態來結合phenylalanine，可造成酵素結構上的改變，而扮演著對酵素異位調控的角色。
從序列的角度看，大部分物種PDT中的ACT domain 具有GALV、ESRP兩段motif，影響著L-Phe的負回饋調控與酵素結構的改變，然而在Buchnera的P-protein中，卻呈現著TSQK的序列，再加上其pheA上游缺乏減弱子，顯示Buchnera的P-protein 並不會受到終產物phenylalanine 的抑制，而可大量合成phenylalanine以提供宿主需求。
因此，我們希望透過電腦輔助藥物設計軟體的幫助，有效尋找PDT的抑制劑，藉由抑制Buchnera中PDT酵素的活性，進而阻斷phenylalanine的生合成，使得宿主蚜蟲無法獲得足夠的必需胺基酸，存活率降低，就可以減少對農業經濟的危害。
在本實驗的研究中，將利用目前僅有的原始 (Native) PDT結構 2QMW (Staphylococcus aureus的PDT，SaPDT) 為模板，透過同源模擬 (Homology model) 建立E. coli及 Buchnera的結構；並採用不同分子對接軟體的評分函數，做為篩選標準；然後從過去文獻中推測可能的活化位，試著將目前已知之抑制劑DNBA及其類似物、受質prephenate，對接到2QMW及做為軟體條件設定之參考，以電腦虛擬篩選的方式，篩選含有十幾萬筆及高度相異性的化合物資料庫IBS，最後經過分子量、評分函數名次、蛋白質分子對接情形、結構具較多親水性基團等條件評估，選出結構相異度較大的20個化合物，將進行in vitro活性實驗，以期能找出具有抑制PDT活性的化合物。

Aphids are insect vectors which transmit virus and damage agricultural economy. Aphids feed strictly on the nutritionally poor diet of phloem sap by piercing-sucking mouthparts, and thus maintain an obligate endosymbiotic association with Buchnera aphidicola, a member of the class -Proteobacteria closely related to Escherichia coli. It is commonly accepted that Buchnera provides essential nutrients to aphids. The enzyme prephenate dehydratase (PDT) is encoded by pheA, and is a terminal and committed enzyme in the biosynthesis of L-Phe in the shikimate pathway. In different organisms, PDTs exist as either monofunctional or multifunctional enzymes. Generally, in Gram-positive bacteria and archaea, PDT is a monofunctional enzyme. However, in Gram-negative bacteria such as E. coli and Buchnera aphidicola APS, PDT exits as a fusion protein with the enzyme chorismate mutase (CM) to form a bifunctional enzyme.
PDT converts prephenate to phenylpyruvate through dehydration and decarboxylation reactions, and is allosterically regulated by the end product L-Phe. The enzyme is only found in microorganisms, fungi, and plants, and is a novel and potential drug target. Hence, we hope to find more effective inhibitors of the Buchnera P-protein through structure-based virtual screening. These inhibitors can be used to reduce the viability of aphids by blocking the growth of Buchnera.
PDT consists of an N-terminal PDT domain (the catalytic domain) and a C-terminal ACT domain (the regulatory domain). The PDT domain contains the substrate (prephenate) binding site. The ACT domain in its dimeric form often binds the end product L-Phe or other amino acids, and produces a conformational change to regulate the activity. Important features of the ACT domain for L-Phe binding are the GALV and ESRP motifs, but in P-protein of Buchnera, the ESRP motif is changed to TSQK. For this reason, P-protein of Buchnera is proposed to be insensitive to feedback inhibition by L-Phe.
In the study, we built the E. coli and Buchnera homology models based on 2QMW, the first and the only native PDT crystal structure. Then, we used different scoring function from the molecular docking program, and analyzed the prephenate, DNBA and their analogs docking results. After the docking program parameter set up, we exercised a virtual screen for PDT inhibitors by docking molecules in IBS chemical databases to 2QMW. In the end, we chose twenty compounds according to their molecular weight, scoring rank, molecular docking pose, hydrophile group and structure heterology. Then, we to test their activity in vitro in the future.

摘要 １
Abstract ２
誌謝 ３
目錄 ４
圖目錄 ６
表目錄 ７

第一章 緒論
1-1 蚜蟲與蚜蟲內共生菌 ８
1-2 芳香族胺基酸合成路徑-莽草酸途徑(Shikimate pathway) ９
1-3 PDT存在的形式 １０
1-4 PDT結構的分析 １０
1-4-1 PDT的功能區(domain) １０
1-4-2 PDT的結構介紹 １１
1-4-3 PDT的序列分析 １２
1-4-3-1 與PDT催化有關的序列 １２
1-4-3-2 與PDT活性抑制有關的序列 １３
1-4-4 PDT的異位調控機制 １４
1-5 PDT相似結構的分析與功能 １５
1-5-1 DALI search of PDT domain １５
1-5-2 DALI search of ACT domain １５
1-6 目前已知PDT的抑制劑 １６
1-7 研究動機與目的 １７

第二章 材料與方法
2-1電腦虛擬篩選（In Silico Virtual Screening〕 １８
2-2分子對接軟體之應用與原理 ２０
2-3評估模擬蛋白質結構的軟體PROCHECK-Ramachandran plot ２２

第三章 策略與結果
3-1 序列比對與活化位的選擇 ２３
3-1-1各物種間的序列比對 ２３
3-1-2活化位的選擇 ２３
3-2軟體的設定 ２４
3-2-1文獻中已做過活性測試的化合物 ２４
3-2-2建立E. coli 與Buchnera aphidicola PDT的模擬結構 ２４
3-2-3 Ligand的繪製 ２５
3-3 篩選策略與分析 ２６
3-3-1初篩介紹 ２６
3-3-2最終化合物的選擇 ２６

第四章 討論與結論
4-1 建立Buchnera aphidicola 與E.coli PDT模擬結構之重要性 ２８
4-2 已知結構2QMW與模擬結構BaPDT分子對接結果之比較 ２８

第五章 未來方向
5-1 進行in vitro實驗 ２９
5-2 建立有效的篩選機制 ２９

第六章 參考文獻
6-1 國內論文 ３１
6-2 國外論文 ３１

圖目錄
Fig1-1-1 植物、宿主蚜蟲與內共生菌Buchnera三者的關係 ３６
Fig1-2-1 PDT的催化作用 ３６
Fig1-2-2 莽草酸途徑 (Shikimate pathway ３６
Fig1-2-3 在E.coli 中，利用Shikimate pathway的中間產物–Chorismate的五種酵素。 ３７
Fig1-3-1. 253個物種建立出的PDT親緣關係樹 (Phylogenetic tree ３７
Fig1-4-1. The ribbon drawing and the scheme of the Sa-PDT structure ３８
Fig1-4-2 PDT family的保守序列。 ３８
Fig1-4-3 Structure-based sequence alignment of PDT domains across different species. ３８
Fig1-4-4 Structure-based sequence alignment of ACT domains of PDT across different species. ３９
Fig1-4-5 Domain organization and reactions of P-protein, PAH and 3PGDH. ３９
Fig1-4-6 The PDT domain dimers and catalytic sites. ３９
Fig1-4-7 The ACT domain dimers and requlatory sites. ４０
Fig1-4-8 The structure of T-state Ct-PDT. ４０
Fig1-4-9 A schematic drawing of the allosteric requlation mechanism of PDT. ４０
Fig1-5-1 PDT domain的類似結構。 ４１
Fig1-5-2 具有ACT domain的3PGDH蛋白質結構。 ４２
Fig1-5-3 具有ACT domain的AHAS蛋白質結構圖。 ４２
Fig1-5-4 具有ACT domain的AKⅢ蛋白質結構圖。 ４３
Fig2-1-1 電腦輔助藥物設計在藥物發展中扮演的角色。 ４４
Fig2-1-2 電腦輔助藥物設計的分類。 ４４
Fig3-1-1 Structure-based sequence alignment of PDT across different species. ４５
Fig3-1-2 Structure-based sequence alignment of PDT across Buchnera、E. coli、two known structure species and three different plants. ４６
Fig3-1-3活化位附近的重要residues。 ４７
Fig3-2-1 目前文獻中已做過PDT活性測試的化合物。 ４８
Fig4-2-1 受質prephenate與模擬結構BaPDT之分子對接結果。 ４９

表目錄
Table 1-6-1.目前已知PDT的抑制劑。 ４９
Table3-1-1 Sequence identity and similarity of PDT across Buchnera、E. coli、Rice and two known structure species. ５０
Table3-2-1 透過分子對接軟體對接含受質prephenate等19個化合物的初步結果。 ５０

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