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標題: | 阿拉伯芥植物金屬螯合素合成酶磷酸化後修飾位點Ser352之研究 Characterization of Ser352 phosphorylation site in Arabidopsis phytochelatin synthase |
作者: | Yan-Hung Chen 陳彥宏 |
指導教授: | 莊榮輝 |
關鍵字: | 植物金屬螯合素,植物金屬螯合素合成?,磷酸化位點Ser352, Phytochelatin,Phytochelatin synthase,Phosphorylation site Ser352, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 植物金屬螯合素合成酶 (phytochelatin synthase, PCS, EC2.3.2.15) 在植物對抗重金屬逆境時扮演著重要的角色。其利用兩種基質glutathione (GSH) 以及和金屬結合的glutathione衍生物 (Cd-GS2),合成出具有金屬結合能力的植物螯合素 (phytochelatin, PC),將重金屬螯合、隔離以降低重金屬對細胞的毒害。真核生物PCS的蛋白質結構可以分為主要催化活性區域的N端,以及含有多個Cys pair、為鎘離子主要結合區的C domain。目前的研究顯示,PCS在生物體內處於持續表現的狀態,但是只有當植物處於重金屬逆境下才能啟動其酵素活性,因此一般認為PCS之活性調節應是屬於轉譯後修飾 (post-translational modification) 之層級。本實驗室先前利用點突變技術以及酵母菌 (Pichia pastoris) 表現系統,發現阿拉伯芥 (Arabidopsis thaliana) AtPCS1 位於C端的Ser352是磷酸化修飾位點之一,對酵素活性也有顯著影響。Ser352在蛋白質一級序列上十分靠近C domain上的金屬結合位Cys358Cys359,本論文發現此磷酸化位點的突變不僅使酵素的活性降低,同時也會造成AtPCS1與金屬結合的比率上升。因此認為Ser352與金屬結合位Cys358Cys359之間可能存在著交互作用。進一步利用酵素動力學分析,發現將AtPCS1上的Ser352突變成Ala後,會使酵素對第一基質與第二基質的親和力大幅提升,但降低其催化速率。目前推測Ser352磷酸化位點會對AtPCS1產生綜合性影響,同時改變酵素對金屬之結合率。另一方面,本研究篩選於阿拉伯芥PCS缺失株cad1-3中分別表現AtPCS1或PCS-S352A之轉殖株。性狀分析結果顯示,4株PCS-S352A轉殖株之鎘耐受度與野生種均無明顯差距。綜合S352A 之生化性質與生理功能分析,本論文推測Ser352磷酸化修飾雖然會影響AtPCS1之酵素功能,但是在生理條件下酵素雖然只保留部分催化活性,但可能可以合成足夠的PC來維持植株在重金屬環境下之生長;而PCS本身也具有螯合重金屬的能力,在植物體內可能是一種優良的重金屬螯合分子,防止重金屬離子對細胞造成毒害。 Phytochelatin synthase (PCS, EC2.3.2.15) plays important roles in the sequestration of heavy metals in Arabidopsis thaliana. It uses two substrates, GSH and Cd-GS2, to synthesize phytochelatin (PC) via ping-pong mechanism. PC chelates heavy metals and forms a high molecular complex, which is then transferred to vacuoles to prevent from damaging the cells. PCS composes of a catalytic domain at N-terminus and a Cys-rich C-terminal domain which is responsive to cadmium (Cd). Many studies show that PCS is constitutively expressed in cytosol but could be only activated under heavy metal stress. The phenomenon indicates that PCS may be regulated by post-translational modification. Our previous data have demonstrated that recombinant AtPCS1 could be activated by in vitro phosphorylation. Furthermore, phosphorylation at Ser352 of recombinant AtPCS1 might affect the catalysis of the enzyme. Ser352 is close to Cys358Cys359, which is a heavy-metal binding site of the C-terminal domain. This study showed that mutation at Ser352 decreased the catalytic activity of PCS while increased the Cd binding ratio of the enzyme. Moreover, Recombinant AtPCS1 with a mutation at Ser352 showed a lower catalytic activity because of the significantly reduced Vmax along with the higher affinities to both substrates, which suggested the role of Ser352 in activation of PCS. On the other hand, this study selected 6 Arabidopsis transgenic lines that express AtPCS1 and AtPCS1-S352A in a PC-deficient mutant, cad1-3. Although S352A has a low catalytic activity in vitro, AtPCS1-S352A transgenic lines showed no significant changes in Cd tolerance comparing to the wild type Arabidopsis. Thus, we speculated that partially-activated PCS could synthesize enough PC to rescue the Cd tolerance of the transgenic lines, and the PCS might also act as a chelator to bind heavy metals in cytosol. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53589 |
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顯示於系所單位: | 生化科技學系 |
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