臺灣博碩士論文加值系統

稻米核苷二磷酸激酶(NDK)的晶體結構
摘要:
核苷二磷酸 (Nucleoside diphosphate kinase ，NDK) 是一普遍存在於各種生物體及各種型態細胞的酵素。它可以催化 NTP (nucleoside triphosphate) 上γ磷酸根的轉移至NDP (nucleoside diphosphate)。在稻米中NDK涉及葉片的延展，並在種子萌芽與早期結實時期發現NDK量的改變。雖然許多菌種及哺乳動物的NDK蛋白結構已經被解出，然而在植物中卻沒有。
實驗中我們從稻米分離出NDK基因的 cDNA，將其轉植入大腸桿菌中使其表現NDK蛋白，純化取得、長晶。在日本Spring8 BL12B2 作X光繞射實驗，得知晶體的空間群為P2 (1)2(1)2(1),單位晶格(unit cell) 為 a=70.98 Å、b=182.26 Å、c=188.30 Å。利用分子替代法解得其相位，並經調至2.52Å解析度，大體的結構為四個摺坂外圍著六個螺旋。從晶體的堆疊與水溶液結構都暗示著NDK以六個分子的形式存在生物體中。稻米NDK結構是第一個植物NDK結構，我們希望這個結構能在植物生長與發育上提供一些資訊。

Crystal Structure of Nucleosie Diphosphate Kinase in Rice
Nucleoside diphosphate kinase (NDK) is a ubiquitous enzyme found in all organisms (eukaryotes and prokaryotes) and cell type. It catalyses the transfer of the tpphosphoryl group from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP). NDK is involved and required for coleoptile elongation in rice. The level of the enzyme changes during seed germination and the early stages of seedling growth. Although several NDK structures from bacteria and animals have previously been available, the structures from plants are not yet determined.
Crystals of the rice NDK have been obtained and several sets of data were collected at SPring-8 BL12B2 in Japan. The crystals belong to the space group P2(1)2(1)2(1) with unit-cell parameters a = 70.98 Å, b = 182.26 Å, c = 188.30 Å, and diffract to 2.5 Å. The structure is determined by molecular replacement method and shows the overall folding with four -sheets surrounded by six -helices. The model contains 149 residues of a total 152 residues and averaged 18 water molecules. Hexameric molecular packing in both crystals and solution implies the rice NDKs function as hexamers. In this study, we have determined the first rice NDK structure, which provides the detailed structural-functional information to understand the functional significance of this enzyme during plants growth and development in rice as well as plants.

第一章 緒論
1-1 核苷二磷酸激酶(NDK)概論
1-2 NDK的三級結構
1-3 NDK的四級結構
1-4 活性區域與機制
1-5 研究目的
第二章 實驗方法與材料
2-1 從植物葉鞘的cDNA中克隆 (Clone) NDK基因
2-2 稻米NDK的表現與純化
2-3 長晶條件與晶體特性
2-4 X光繞射數據的收集與處理
2-5 結構分析方法
2-6 結構精調
2-7 色層分析
第三章 實驗結果與討論
3-1 稻米NDK的結構
3-2 稻米NDK與其他物種活性區的比較
3-3 稻米NDK 四級結構的探討
3-4 稻米NDK晶體堆疊的探討
第四章 參考資料
附圖表

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