阿拉伯芥植物螯合素合成酶C端區塊功能之研究

The role of the cysteine-rich C terminal domain of phytochelatin synthase in Arabidopsis thaliana

陳怡靜(Yi-Jing Chen)

指導教授 ： 莊榮輝

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00851

摘要

阿拉伯芥 (Arabidopsis thaliana) 植物螯合素合成酶 (phytochelatin synthase, PCS, EC 2.3.2.15) 利用 glutathione (GSH) 為基質，合成可和金屬結合的植物螯合素 (phytochelatins, PC)，以降低重金屬對植物的危害。真核生物的 PCS 在蛋白質結構上分成主要催化區的 N 端 domain，以及含有多個成對 Cys 的 C 端domain。本論文以平衡透析法確認 AtPCS1 的 C 端 domain 為主要的金屬結合區，除了已知的 Cys358Cys359 外，也找到另一個金屬結合 Cys342Cys343。為了瞭解 C 端 domain 之金屬結合位與鎘離子結合後對 PCS 構形的影響，首先製備 A site 突變株 (C342S, C343S)、B site 突變株 (C358S, C359S)、C site 突變株 (C363S, C366S)、B+C site 突變株 (C358S, C359S, C363S, C366S) 和 A+B+C site 突變株 (C342S, C343S, C358S, C359S, C363S, C366S)，以 tryptophan 螢光光譜檢測，發現 wild type 和鎘結合後會導致螢光下降約 28%。然而突變株在結合鎘離子後，tryptophan螢光光譜與 wild type 有很大的不同，表示鎘離子和 PCS 結合可能導致蛋白質構形改變。本論文也以高通量即時螢光定量 PCR 系統測量蛋白質的 Tm 發現 wild type 與鎘結合後蛋白質穩定度會上升；相反地，C 端金屬結合位突變株在相同鎘離子濃度下之穩定度會下降。另一方面，要確認 C 端金屬結合位突變株對活性的影響，利用硫醇基 (thiol group) 上帶有甲基化修飾的 S-methylglutathione為基質進行活性分析，結果發現加入 0.5 µM 鎘可提升 wild type PCS 的催化活性，但對 C 端金屬結合位突變株的活性並沒有明顯影響。進一步以 GSH 為基質進行酵素動力學檢定，發現金屬結合位突變株之 Vmax/Km 比 wild type 低，顯示突變株的整體催化能力下降，由此結果推測鎘離子藉由結合到 C 端金屬結合位上影響 PCS 催化活性。綜合以上結果推測鎘與 PCS 上的 Cys motif 結合後，可能會造成蛋白質構形改變，進而影響 PCS 的整體催化活性。

關鍵字

阿拉伯芥 ； 植物螯合素 ； 植物螯合素合成酶重金屬 ； 金屬結合位

並列摘要

Phytochelatin synthase (PCS, EC 2.3.2.15) in Arabidopsis thaliana utilizes glutathione (GSH) as the substrate to synthesize phytochelatins (PC) which are high-affinity metal chelators. PCS has a highly-conserved N-terminal domain which contains the catalytic triad, and a variable Cys-rich C-terminal region which might involve in heavy metal binding. In this study, we demonstrated that the C-terminal domain was the major metal-binding region on AtPCS1, and identified a new metal binding motif, Cys342Cys343. To explore the function of the metal-binding sites on C-terminal domain, the C-terminal mutants were made containing A site mutant (C342S, C343S), B site mutant (C358S, C359S), C site mutant (C363S, C366S), B+C site mutant (C358S, C359S, C363S, C366S) and A+B+C site mutant (C342S, C343S, C358S, C359S, C363S, C366S). The tryptophan fluorescence spectrum of cadmium-bound PCS showed declined fluorescence levels indicating that bound metals might cause the conformation changes of the protein. Cadmium binding could also increase the stability of the protein and resulted in a raised Tm value. However, the tryptophan fluorescence spectra of Cys mutants were significantly different than wild type, which might have distinct metal-response. After incubated with cadmium, the Cys mutants showed declined in Tm values, which indicated that loss of the metal binding sites might cause unstable structures of the proteins. Besides, the catalytic activity of the Cys mutated PCS using S-methylglutathione as substrates was analyzed. The S-methyl-PC synthesis of wild type PCS was activated by cadmium, while the presence of cadmium could not elevate the activity of the mutants. Moreover, the overall catalytic activity of the Cys mutants was much lower than wild type. These results suggested that PC synthesis might be influenced by cadmium binding on the metal binding motif. Taken together, we proposed that the Cys motif might act as the Cd sensor on AtPCS1, and the interaction between Cd and the Cys motifs might lead to the conformational change and change the catalytic activity of the enzyme.

並列關鍵字

Arabidopsis thaliana ； phytochelatin ； phytochelatin synthase ； heavy metal ； metal binding site

參考文獻

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