指導教授:莊榮輝臺灣大學:生化科技學系賈儒珍Chia, Ju-ChenJu-ChenChia2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261605Phytochelatin synthase (PCS) uses the substrates glutathione (GSH, γGlu-Cys-Gly) and a cadmium (Cd)-bound GSH (Cd∙GS2) to produce the shortest phytochelatin product (PC2, (γGlu-Cys)2-Gly) through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12–218) by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS) as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation) resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W) for the Cd∙GS2, further suggesting the role of the cation-πinteraction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed. Besides the effects of metal ions on substrate binding mechanism, the post-translational activation of PCS via phosphorylation was also discussed in this study. Previous studies had revealed that Thr49 is the site for phosphorylation and play an important role in the formation of the active site through the interaction with Arg183. In addition, Ser13 and Ser352 were also identified as phosphorylation sites of AtPCS1 expressed in Pichia pastoris expression system. In contrast with the activity of T49A mutant, mutations on these Ser residues did not significantly affect PCn catalysis of PCS. Ser352 was located on the heavy metal binding motifs on C-terminal domain, and phosphorylation on this site might involve in the metal-binding ability but not directly influence the catalysis of the enzyme. Besides, these Ser phosphorylation sites showed their identity in Arabidopsis. In the modest cadmium concentration, wild type Arabidopsis and PCS1 complementary lines were more tolerant than S13A and S532A lines, indicating that the phosphorylation sites on AtPCS1 are important for cadmium resistance of Arabidopsis. In summary, our data demonstrated that PCS activity is regulated via post-translational modification, and phosphorylated AtPCS1 may play critical roles in heavy metal stress in Arabidopsis.中文摘要 iii Abstract v 縮寫表 vii 第一章 緒論 1 1.1 重金屬與環境 1 1.1.1 重金屬的定義 1 1.1.2 重金屬之毒害 1 1.1.3 清除重金屬的方法 2 1.1.4 植物金屬螯合素 2 1.2 植物螯合素合成酶之特性 3 1.2.1 植物螯合素合成酶基因家族 3 1.2.2 植物螯合素合成酶之生理功能 4 1.2.3 植物螯合素合成酶轉殖株之性狀 5 1.2.4 植物螯合素合成酶之表現與調控 6 1.3 植物螯合素合成酶之活性催化機制 8 1.3.1 植物螯合素合成酶之蛋白質構造 8 1.3.2 植物螯合素的合成機制 9 1.3.3 植物螯合素合成酶的基質結合區 10 1.4 研究動機 11 第二章 材料與方法 13 2.1 材料 13 2.1.1 菌株 13 2.1.2 植物材料 13 2.2 載體之構築 13 2.2.1點突變技術 (site-direct mutagenesis) 13 2.2.2 P. pastoris表現系統之載體構築 14 2.2.3 阿拉伯芥轉殖載體之構築 14 2.3 重組蛋白質之表現與純化 15 2.3.1 E. coli表現系統 15 2.3.2 P. pastoris表現系統 15 2.3.3 表現蛋白質之純化 16 2.3.4 表現蛋白質之二維電泳 (2-dimensional electrophoresis) 16 2.4 PCS活性分析 17 2.4.1 PCS活性分析及活性單位之計算 17 2.4.2 酵素動力學分析 18 2.4.3 表現蛋白質去磷酸化處理 18 2.5 阿拉伯芥轉殖 18 2.5.1 農桿菌轉殖法 18 2.5.2 轉殖株篩選 19 2.5.3 RNA之抽取及半定量RT-PCR 19 2.5.4 Western blot 20 2.6 阿拉伯芥鎘逆境處理及性狀分析 20 2.7 阿拉伯芥內生性AtPCS1之純化 20 2.8 其他分析方法 21 2.8.1 AtPCS1鎘結合能力之測定 21 2.8.2 AtPCS1分子模型之建立 21 2.8.3 蛋白質身分及轉譯後修飾之鑑定 22 2.8.4 Pro-Q diamond染色法 22 第三章 結果與討論 27 3.1 PCS序列和結構分析 27 3.1.1 植物螯合素合成酶胺基酸序列比對 27 3.1.2 AtPCS1分子模型之建立 27 3.2 AtPCS1第二基質結合位之研究 37 3.2.1 第二基質結合區之重要胺基酸 37 3.2.2 Tyr55可能與第二基質上的金屬離子結合 42 3.2.3 AtPCS1第二基質結合區之分子機制 51 3.3 AtPCS1之磷酸化後修飾 54 3.3.1 以酵母菌系統表現AtPCS1 54 3.3.2 以P. pastoris生產的AtPCS1有磷酸化現象 57 3.3.3 AtPCS1的磷酸化修飾位置 61 3.3.4 Ser13和Ser352磷酸化修飾位置序列分析 67 3.3.5 Ser13和Ser352磷酸化會影響蛋白質活性 69 3.3.6 阿拉伯芥AtPCS1-S13A、S352A補償性轉殖株之建立 72 3.3.7 阿拉伯芥AtPCS1-S13A、S352A補償性轉殖株之基因表現 80 3.3.8 AtPCS1磷酸化位置突變會影響阿拉伯芥對鎘之耐受性 82 3.4 阿拉伯芥內生性PCS之純化 87 3.4.1部分純化阿拉伯芥內生性PCS 87 3.4.2 LC-MS/MS身份鑑定 88 第四章 總結 95 參考文獻 97 問答錄 1054969492 bytesapplication/pdf論文公開時間:2024/02/14論文使用權限:同意無償授權植物螯合素植物螯合素合成酶酵素催化機制阿拉伯芥轉譯後修飾重金屬逆境阿拉伯芥植物螯合素合成酶第二基質結合位及轉譯後修飾之研究Characterization of the Second Substrate Binding Site and Post-Transpational Modification in Arabidopsis Phytochelatin Synthasethesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261605/1/ntu-103-D96b47202-1.pdf