甘草甜素及阿拉伯芥中細胞色素P450 氫氧化酶之分子選殖、蛋白質表現及其功能性分析

Abstract

皂苷其結構是由親脂性的三萜類單元以及親水性的醣基單元所組成。據先前 文獻報告指出，皂苷大多擁有生物活性，例如像是抗發炎、抗菌、抗微生物或是 抗癌作用。而就皂苷的生合成途徑而言，其結構衍生化主要可分為三階段: (1) 經 由氧化鯊烯環化酵素家族所產生的多樣性三萜類骨架﹔（2）經由氫氧化酶或是 氧化酶所進行的第一階段後修飾作用﹔ (3)利用醣基轉移酶在氫氧基上接上醣 基而完成的最終皂苷產物。而本實驗室在先前的研究中，針對酵母菌之氧化鯊烯 環化酵素其環化作用進行「結構-功能-作用機制」的分析，並獲得數個在環化機 制中扮演重要角色的胺基酸位置，進而分離出多個在環化/重組過程中的關鍵性 中間產物結構。為了利用這些多樣性的三萜類物質進行其骨架修飾，以增加其結 構多樣性，並使其能應用於下游的皂苷衍生化之酵素反應或更進一步的形成製藥 前驅物。扮演關鍵性的第一階段修飾酵素-細胞色素P450 氫氧化酶則成為重要的 研究課題。而在此論文中，我們針對三個具有三萜類後修飾功能之細胞色素P450 氫氧化酶進行其分子轉殖及蛋白表現並藉由活性測試對於表現的蛋白進行初步 的功能性鑑定。我們分別選用了甘草甜素中的CYP88D6 蛋白及阿拉伯芥中的 CYP90B1 及CYP90D1 蛋白，並成功地接合所有的人造基因於多個表現載體中。 雖然大部分的表現系統皆無法有效地產生重組蛋白質，但pRSETa-CYP88D6 及 pET30a-CYP90B1 質體最後可以成功地在大腸桿菌系統E.coli BL21(DE3)pLysS 中進行其重組蛋白的表現，並且利用基質輔助雷射吸附質譜技術鑑定出此這兩個 重組蛋白為分別為氫氧化酶CYP88D6 及CYP90B1。而在活性測試的部份， CYP88D6 蛋白對於β-香桂脂醇 以及CYP90B1 蛋白對於菜油固醇都具有催化能 力，並能在氣相質譜儀上產生氫氧基衍生化產物的訊號。此外我們利用Ni-NTA 樹脂凝膠或 Q-sepharose 離子性樹脂凝膠進行初步的蛋白質純化，並藉由SDS 電泳分析其蛋白質純度，與利用紫外光-可見光光譜儀獲得其特性化的Soret band吸收頻譜，這些實驗結果能進一步的支持我們純化表現的重組蛋白分別為甘草甜素中的CYP88D6 蛋白及阿拉伯芥中的CYP90B1。目前我們對於蛋白質生產過程與純化過程進行改造以獲得更大量的純化蛋白以期能運用於活性機轉的探討。此外藉由突變效應以進行酵素產物專一性與酵素活性的改良也是一個引人注目的 方向，我們希望在未來，能利用這些改良的氫氧化酶針對我們所獲得的多樣化三 萜類產物進行衍生化，使其帶有不同的生理功能以祈能應用於未來藥物學方面的 研究。

Triterpene saponins constitute a family of compounds with a triterpene aglycone and sugars structures. They were widely distributed in various species such as food, echinoderm, fungi and plants. Recent researches have demonstrated that triterpene saponins exhibited different biological activities from antiinflammatory, antifungal, antimicrobial, antiparasitic, and antitumor activity. Biosynthetic studies suggested that the structure diversity of triterpene saponin is generated by (1) the cyclization of oxidosqualene to various triterpene skeletons, (2) the subsequent hydroxylation or oxidation at multiple positions of triterpene, and (3) glycosylation to add sugar moieties through an ether or ester glycoside linkage. Based on our previous investigations on the S. cerevisiae oxidosqualene-lanosterol cyclase (ERG7), we have identified several particular amino acid residues associated with the cyclization and acquired various novel intermediated structures via proceeding with mutation on OSC. According to the biosynthesis of triterpene saponins, the first modification, followed by triterpene cyclization, is hydroxylation. In order to further diversify the scaffold of triterpene saponins for the potential pharmaceutical applications, in this thesis, we cloned and functionally expressed three hydroxylases, CYP88D6 (Glycyrrhiza uralensis, C-11 hydroxylase), CYP90B1 (Arabidopsis thaliana, C-22 hydroxylase), and CYP90D1 (Arabidopsis thaliana, C-23 hydroxylase) for activity study. Various expression systems including E. coli or yeast, coupled with a series of expression vectors, have been used to clone these artificial genes successfully. Even most of them failed to produce the recombinant proteins as predicted, the CYP88D6 from pRSETa-CYP88D6 plasmid and CYP90B1 from pET30a-CYP90B1 plasmid were over-expressed in E.coli BL21(DE3)pLysS strain and were confirmed the protein identity by MALDI-TOF/MS spectrum. Moreover, we have examined the potential hydroxylation activity of CYP88D6 and CYP90B1 by using the reconstituted assay with human NADPH-cytochrome P450 reductase and NADPH. The microsome fraction from E. coli expressing G. uralensis CYP88D6 or A. thaliana CYP90B1 exhibited their catalysis ability in transforming a hydroxyl group into the scaffold of the substrates, β-amyrin or campesterol, to form the respective hydroxyl products with predicted molecular mass from the ion extraction of GC–MS analysis, implying a functional activity of expressed cytochrome P450 hydroxylase, respectively. The partial purification of G. uralensis CYP88D6 or A. thaliana CYP90B1 were carried out via Ni-NTA column purification or Q-sepharose purification, and characterized by SDS-PAGE electrophoresis as well as UV-vis spectrum analysis at its Soret band absorption at 416 nm. Various modifications on the production and purification of functional cytochrome P450 hydroxylase are on the process in hope of acquiring the large scale of purified protein for studying their reaction mechanism and being subjected for sterol compounds’ tailoring reactions to produce various sterol derivatives, that carrying various biological activities to be applied in pharmacology. Moreover, mutagenic effect on product specificity as well as on the reaction efficiency will also be explored for tuning the expressed cytochrome P450 hydroxylase in future.