阿拉伯芥金屬螯合素合成酶轉殖株之分子鑑定及Thr49突變株之活性分析

Abstract

金屬螯合素合成酶 (phytochelatin synthase, PCS) 之活性調控方式，可能經由轉譯後之修飾 (post-translational modification) 而非轉錄層次。以蛋白質磷酸化預測軟體分析阿拉伯芥PCS1序列，發現Thr49附近有很強烈的CK2磷酸化訊號。進一步以藍綠藻NsPCS立體構造為模版，建立AtPCS1的N端區域三級結構，發現Thr49在立體結構上非常靠近Arg183，因此推論Thr49可能藉由磷酸化與Arg183的正電基團產生離子鍵作用力，造成構型改變進而提升PCS的催化活性。由蛋白質定位點突變實驗，已證實PCS的活性確實以磷酸化及去磷酸化調控。本論文針對PCS和三種Thr49點突變株T49A、T49D、T49E進行酵素動力學分析，結果發現T49A的Kcat明顯下降為wild-type (WT) 的23%，T49D和T49E也分別只有WT的7.6% 和10.2%；另外，T49A的Km值是WT的69%，而T49D和T49E分別為WT的5.8倍和1.9倍。由此可知，當Thr49置換成酸性胺基酸後，對於PCS的基質結合力和催化效率都與動機之推測相反。PCS可能以可逆的磷酸化調控來改變酵素的構形，Thr49上的磷酸化能穩定活性區的立體結構，進而提升PCS的催化效率。此外，由於植物內的PCS表現量很少，為了純化出大量的內生性PCS，我們將AtPCS1在阿拉伯芥中過量表現，經由抗生素篩選、PCR、半定量RT-PCR及IPCR等分子檢測，得到穩定表現AtPCS1之T3代轉殖株，期望藉由提高植物內PCS蛋白質表現量，純化得內生性的PCS，以進而深入研究PCS之磷酸化在植株內的生理角色。

AtPCS1 is constitutively expressed in the Arabidopsis. Its mRNA level is not enhanced by the pretreatment of plant with heavy metals. This implies that AtPCS1 could be controlled by post-translational modification; and protein phosphorylation is one of the essential modification mechanisms in the cell. From the amino acid sequence of AtPCS1, Thr49 was predicted to be a potential phosphorylation site. The three-dimensional structure of NsPCS was used as the template for the computer modeling of the N-terminal domain of PCS from Arabidopsis. It was found that the side chain of Arg183 was very close to the phosphate group of the phospho-Thr49, and interaction between this phosphorylated group and the Arg183 may form a functional conformation for the catalytic site of PCS. Pre-vious studies have shown that phosphorylation status may affect PCS activity. In this study, several site-directed mutants on Thr49 were performed including AtPCS1(T49A), AtPCS1(T49D) and AtPCS1(T49E). After expression of the mutant proteins, we examined the kinetic parameters of PCS mutants. The Kcat of mutant proteins T49A, T49D and T49E decreased to 23%, 7.6% and 10.2% of the wild type protein, respectively. On the other hand, the Km of T49A decreased to 69%, but T49D and T49E had 5.8-fold and 1.9-fold increase in Km. Apparently, when Thr49 was replaced by acidic amino acid, the substrate affinity and catalytic efficiency of PCS was inhibited. It was postulated that PCS activity might be controlled by reversible phosphorylation, and the conformation of the enzyme can be changed by the introduction of this phosphate group. This conformation change might facilitate the entrance of the substrate into the catalytic sites. In addition, to purify the endogenous PCS from the plant, we tried to overexpress AtPCS1 in Arabidopsis. After antibiotic selection and the molecular identification of AtPCS1 by PCR, semi-quantitative RT-PCR and IPCR, we might obtain the transgenic Arabidopsis producing larger amount of PCS for further studies of the protein phosphorylation of the endogenous PCS.