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標題: | "紫茉莉中參與甜菜⾊素⽣合成之4,5-多巴雙加氧酶的結構與功能探討" Functional and structural studies of a 4,5-DOPA dioxygenase involved in betalain pigment biosynthesis from Mirabilis jalapa |
作者: | Yin-Chia Chou 鄒穎佳 |
指導教授: | 徐駿森(Chun-Hua Hsu) |
關鍵字: | 甜菜?素,甜菜醛氨酸,外雙加氧?,紫茉莉,植物?素,晶體結構, Betalains,betalamic acid,extradiol dioxygenase,Mairabilis jalapa,plant pigment,crystal structure, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 甜菜色素是一含氮的水溶性色素,存在於石竹目(Caryophyllales) 植物的花、果實或其他部位。甜菜醛胺酸是構成天然甜菜色素的基本單元,以左多巴 (3,4-dihydroxy-L-phenylalanine , L-DOPA) 作為前驅物,由關鍵酵素4,5-多巴雙加氧酶 (4,5-DOPA-extradiol dioxygenase) 進行催化生成中間產物 4,5-seco-dopa,在經過自發性分子內環化最後生成甜菜醛胺酸。本論文對紫茉莉 (Mairabilis jalapa) 的4,5-多巴雙加氧酶 (簡稱mjDOD) 進行相關功能與結構特性探討,圓二色光譜儀顯示mjDOD具有良好的熱穩定性,Tm約為 58°C,在pH 3.5 ~ pH 8.5的環境中都可以維持正確折疊,而其活性最適pH值為6.0。此外,mjDOD之晶體結構亦被解構至解析度為2.66 Å,結構顯示mjDOD 之催化中心由 H9、H47 和 H222 與二價鐵離子形成配位,推測此酵素可利用非血基質鐵 (non-heme Fe(II)) 催化開環反應,因其催化中心結構特徵與其他外雙加氧酶相似。在催化中心的附近有兩個具有保守性的胺基酸 H112 和 H168 ,對其做點突變後會使酵素完全失去活性,表示這兩個胺基酸在催化反應中扮演重要角色。根據這些結果,我們提出 L-DOPA 與 mjDOD 結合的可能模式與酵素催化機制。據我們所知,此mjDOD的晶體結構為第一個被解析的植物類型4,5-多巴雙加氧酶結構。 Betalains are water-soluble nitrogenous pigments, synthesized in flowers, fruits and other tissues of the plant order Caryophyllales. Betalamic acid is the structural unit of natural pigments betalains. The formation of betalamic acid from the precursor amino acid L-DOPA (3,4-dihydroxy-L-phenylalanine) is catalyzed by the key enzyme 4,5-DOPA-dioxygenase followed by intramolecular cyclization of the 4,5-seco-dopa intermediate. In this thesis, we examined the functional properties and structural characteristics of a 4,5-DOPA-dioxygenase (mjDOD) from Mairabilis jalapa. Circular dichroism spectra showed that mjDOD has good thermal stability with the Tm value as 58°C, and can maintain a correct fold at pH range of 3.5 to 8.5. The pH optimum for enzyme activity toward L-DOPA was shown to pH 6.0. In addition, the crystal structure of mjDOD was determined at 2.66 Å resolution, showing that the Fe-ligand residues are H9, H47 and H222 and indicating that the enzyme uses non-heme Fe(II) to catalyzing a ring opening reaction, which also found in many other extradiol dioxygenase. Since two conserved residues H112 and H168 are observed near the active site, site-directed mutagenesis was conducted for further examination. Interestingly, the mutants are totally inactive. According to these results, the substrate binding mode and catalytic mechanism of mjDOD are proposed. To the best of our knowledge, the crystal structure of mjDOD is the first solved plant 4,5-DOPA-dioxygenase. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68589 |
DOI: | 10.6342/NTU201703523 |
全文授權: | 有償授權 |
顯示於系所單位: | 農業化學系 |
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