氧化鯊烯環化酵素之易曲性及其於形成具生物活性產物之研究

Abstract

氧化鯊烯環化酵素之易曲性及其於形成具生物活性產物之研究 氧化鯊烯為黴菌中麥角脂醇、哺乳動物之膽固醇、及植物中之植物脂醇等生合成途 徑中最後一個共同之中間產物。氧化鯊烯環化酵素催化直鏈之氧化鯊烯之環化及重組反 應形成具多個立體中心之多環產物如四環之羊毛脂醇和環阿屯醇、五環之-香桂素和羽 扇醇，以及植物之二級代謝產物。針對氧化鯊烯環化酵素所催化反應之產物多樣性及反 應機制之複雜性，以及其可能作為發展降膽固醇、抗細菌、抗黴菌、及抗癌藥物標的之 潛力，已吸引科學家對其投入超過半個世紀以上時間之研究。 本實驗室利用基因遺傳工程技術以及產物分離和鑑定等策略對酵母菌之氧化鯊烯環 化酵素所催化之環化重組反應之結構-功能-作用機制關係有初步之了解。我們在過去以 來，已確認數個對環化或重組過程中扮演重要角色之胺基酸，並由氧化鯊烯環化酵素之 變種中分離數個在環化或重組過程中被中斷之中間產物。這些結果支持酵素之結構-功 能-作用機制關係以及提供作為未來藥物應用之基礎。雖然截至目前所獲得之結果甚為 樂觀，但若能對蛋白質之可塑性及所產生產物專一性或多樣性之過程有進一步之了解， 將可對將來將此類在氧化鯊烯環化過程之中間產物應用於降膽固醇或抗黴菌藥物之開 發有所幫助。另外若在所產生之中間產物，利用三萜類生合成之修飾酵素進行官能基或 衍生物之修飾，將可增加其結構之多樣性以及幫助其生理活性。 為了達到上述之目標，我們將繼續探討酵母菌之氧化鯊烯環化酵素之結構-功能-作用 機制關係，研究蛋白質之混雜性，及設計具新功能之酵素。同時我們也將選殖及表現三 萜類生合成途徑之修飾酵素如氫氧化酵素、醣基轉移酵素、及醯基轉移酵素。經由酵素 修飾作用將可增加所獲得三萜類中間產物之結構多樣性。所修飾之三萜類中間產物將用 於抗菌及抗癌藥物之活性測試。 因此，本計畫在未來三年之工作目標將進行以下數項之工作: (1) 針對氧化鯊烯環化酵素之可能進行分歧演化之區域進行蛋白質工程研究。 A. 氧化鯊烯環化酵素之分歧演化之區域之生物資訊分析。 B. 氧化鯊烯環化酵素之分歧演化之區域之蛋白質工程。 C. 利用2,3:22,23-雙氧化鯊烯做為可能之受質以增加其官能基之可行性。 D. 利用GC-MS 及LC-MS 分析所產生之截短或神奇之產物。 (2) 分子選殖及蛋白質表現三萜類生合成途徑之修飾酵素及裁製修飾截短或神奇之 產物。 A. 分子選殖及蛋白質表現三萜類氫氧化酵素及裁製修飾截短或神奇之產物。 B. 分子選殖及蛋白質表現三萜類醣基轉移酵素及裁製修飾截短或神奇之產物。 C. 分子選殖及蛋白質表現三萜類醯基轉移酵素及裁製修飾截短或神奇之產物。 (3) 三萜類新產物之抗腫瘤及抗細菌和抗黴菌生理活性分析。 A. 細胞培養準備以進行藥物效應分析。 B. 評估所產生之截短或神奇之產物或其裁製修飾物之細胞毒性。 C. 評估所產生之截短或神奇之產物或其裁製修飾物對細胞增生及程式性凋亡之 影響。 D. 評估所產生之截短或神奇之產物或其裁製修飾物於程式性凋亡過程之形態變 化。 E. 評估所產生之截短或神奇之產物或其裁製修飾物於DNA 斷裂之分析。 F. 評估所產生之截短或神奇之產物或其裁製修飾物於抗菌活性之分析。 G. 評估所產生之截短或神奇之產物或其裁製修飾物於抗黴菌活性之分析。 本計畫之執行，對於氧化鯊烯環化酵素之結構-功能-作用機制之關係將可有進一步之 了解，並可能產生具新功能或能產生新產物之新酵素，增加所產生截短或神奇之產物或 其裁製修飾物之結構多樣性，以及提供作為未來獲得具氧化鯊烯環化酵素抑制活性之抑 制劑及其作為抗細菌、抗黴菌、及抗癌藥物之應用。

The Research on Oxidosqualene Cyclase Enzyme Promiscuity and Its Application in Bioactive Components Formation (3S)-2,3-oxidosqualene is the last common intermediate involved in the biosynthesis of ergosterol in fungi, cholesterol in mammals, and phytosterols in higher plants. The oxidosqualene cyclase enzymes catalyze the cyclization/rearrangement of acyclic oxidosqualene into stereochemically rich polycyclic products such as tetracyclic lanosterol and cycloartenol as well as pentacyclic -amyrin, lupenol, and plant secondary metabolites. The product diversity and mechanistic complexity of oxidosqualene cyclase-catalyzed reactions and its potential application in developing hypocholesterolemic, antimicrobial, antifungal, and anticancer drugs have fascinated scientists for over a half century. We have applied genetic engineering coupled with product characterization to elucidate the structure–function-reaction mechanism relationships of the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase-catalyzed (ERG7) cyclization/rearrangement reaction. Several amino acids involved in the catalysis have been identified. In parallel, isolation of truncated or altered deprotonation products produced by the ERG7 mutants directly supports the structure–function-reaction mechanism relationships and provides basis for the pharmaceutical applications. Although preliminary results obtained up-to-date are promising, further detailed understanding of the protein plasticity and product diversity /specificity is prerequisite for successful development of lanosterol-type hypocholesteremic and antifungal or anticancer drugs. In addition, modification of lanosterol-type core structures with tailored enzymes holds great potential both in increasing structure diversity and in enhancing biological activity. In order to advance the abovementioned goals, we will continue to explore the structure-function-reaction mechanism relationships of ERG7, to elucidate the promiscuity of the enzyme and to engineer ERG7 with new activity. In parallel, the isolated truncated or altered products will be subjected to modification with tailored enzymes involved in triterpenoid biosynthesis in order to increase structure diversity and biological activity. Tailored enzymes involved in triterpenoid biosynthesis such as hydroxylase, glycosyltransferase, and acyltransferase will be cloned, overexpressed, and used to carry out further structural modifications. Finally, the biological activity of the modified triterpenes will be evaluated for biological function on antitumor and antimicrobial activities. For the next three years, the research direction will focus on: (1) Protein engineering of oxidosqualene cyclases with designed divergent evolution properties. A. Bioinformatic analysis and homology structural modeling of various oxidosqualene cyclases for designed divergent evolution. B. Protein engineering of cyclization biosynthesis specific regions for designed divergent evolution. C. Evaluation of 2,3:22,23-dixoidosqualene as substrate of oxidosqualene-lanosterol cyclase Mutants. D. Identification of novel triterpenes using GC-MS and LC-MS analyses. (2) Molecular cloning, functional expression of triterpenoid biosynthesis tailoring enzymes, and enzymatic modification of truncated or altered or novel new products. A. Molecular cloning and functional expression of triterpene hydroxylases. B. Molecular cloning and functional expression of triterpene glycosyltransferases. C. Molecular cloning and functional expression of triterpene acyltransferases. (3) Bioactivity Assay of tailored lanosterol-truncated components on antitumor and antimicrobial activity. A. Preparation of cell culture for drug effect evaluation. B. Evaluation of putative bioactive components on cell cytotoxicity. C. Evaluation of putative bioactive components on different cell proliferation or apoptosis. D. Evaluation of drug effect of putative bioactive components on different cell apoptosis through morphological studies. E. Evaluation of putative bioactive components on different cell through DNA fragmentation assay. F. Evaluation of putative bioactive components on antimicrobial activity. G. In vitro antifungal activity assay. Therefore, the execution of this project will advance the understanding of the structure-function-reaction mechanism relationships of OSC enzyme, redesign OSC enzyme with new functions, increase truncated or altered product diversity, and extend potential for obtaining of OSC inhibitor for antifungal, hypocholesteremic, or anticancer drug applications.