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研究生:張育瑞
研究生(外文):Yu-Jui Chang
論文名稱:枯草芽孢桿菌鳥糞嘌呤去胺酶之選殖、純化、酵素特性分析與晶體結構測定
論文名稱(外文):Cloning, Isolation, Characterization, Crystallization and Structural Determination of Bacillus subtilis Guanine Deaminase
指導教授:廖淑惠廖淑惠引用關係張若南張若南引用關係
指導教授(外文):Shwu-Huey LiawZo-Nan Chang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:鳥糞嘌呤去胺酶胞嘧啶核苷去胺酶超家族結構測定
外文關鍵詞:Guanine Deaminasecytidine deaminase superfamilyStructural Determinationdomain swapping
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  • 被引用被引用:4
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Guanine Deaminase (GD, EC 3.5.4.3) 可對guanine進行去胺反應,並將之催化成xanthine,是核酸新陳代謝的重要酵素之一。本篇論文先由大腸桿菌表現系統得到高純度的Bacillus subtilis GD (bGD) 重組蛋白,進行酵素活性分析後得知其Km和kcat分別為56 μM和20.8 s-1。接著成功地培養出bGD蛋白晶體,以X光照射晶體後得到繞射點,利用Se-MAD的方法測定出蛋白結構,解析度為1.15 Å。本結構屬於cytidine deaminase (CDA) fold,其較特殊處是兩單體的domain-swapped C端,由helices αD1與αD2延伸至對方的活性區,且其末端的殘基 (Tyr 156) 直接與受質作用,使兩單體互相纏繞而形成緊密的二聚體結構,以3900 Å2的面積互相接觸。在無外加任何金屬離子的情形下,仍有一鋅離子緊密結合於活性區,與His53,Cys83,Cys86和一個水分子形成四面體鍵結 (tetrahedral coordination)。根據活性區所結合的imidazole和水分子,可模擬反應中間產物與bGD之交互作用,以說明bGD之催化機制和受質的辨識。此晶體結構為封閉構型 (closed conformation),顯示了當受質進入活性區時,C端尾部會與受質作用,因而覆蓋住活性區。所以對於bGD而言,C端的domain swapping不但可幫助二聚體的形成並提升結構的穩定度,而且還可誘導辨識受質的殘基在正確的空間位置,以達受質特異性。與其他CDA superfamily成員的結構比較,顯示了這些成員結構之相同與相異處,蛋白結構的可塑性雕塑了蛋白功能的多樣性。這些結構的比較,幫助了我們預測此超家族尚未解出結構之蛋白成員,其可能參與受質辨識之殘基。
Guanine deaminase (GD), a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. In this thesis, the 156-residue GD from Bacillus subtilis was first cloned, expressed and isolated. The Km and kcat value is 56 μM and 20.8 s-1. The crystal structure has been solved at 1.15 Å resolution by the Se-MAD method. Unexpectedly, the C-terminal segment is swapped to form an inter-subunit active site and an intertwined dimer with an extensive interface of 3900 Å2 per monomer. The essential zinc ion is ligated by a water molecule together with His53, Cys83 and Cys86. The reaction intermediate was modeled into the active-site cavity based on the tightly bound imidazole and water molecules, showing the conserved deamination mechanism and the specific substrate recognition by Asp114 and Tyr156’. The closed conformation reveals that substrate binding seals the active-site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping in GD contributes not only for the oligomerization, the structural stability, but also for the substrate specificity. Finally, structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites in guanine, cytosine and cytidine deaminases, and suggests putative specific substrate-interacting residues for other members such as the dCMP deaminase.
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