标题: | 氧化鲨烯环化酵素之易曲性及其于形成具生物活性产物之研究 The Research on Oxidosqualene Cyclase Enzyme Promiscuity and Its Application in Bioactive Components Formation |
作者: | 吴东昆 WU TUNG-KUNG 国立交通大学生物科技学系(所) |
公开日期: | 2012 |
摘要: | 氧化鲨烯环化酵素之易曲性及其于形成具生物活性产物之研究 氧化鲨烯为霉菌中麦角脂醇、哺乳动物之胆固醇、及植物中之植物脂醇等生合成途 径中最后一个共同之中间产物。氧化鲨烯环化酵素催化直链之氧化鲨烯之环化及重组反 应形成具多个立体中心之多环产物如四环之羊毛脂醇和环阿屯醇、五环之-香桂素和羽 扇醇,以及植物之二级代谢产物。针对氧化鲨烯环化酵素所催化反应之产物多样性及反 应机制之复杂性,以及其可能作为发展降胆固醇、抗细菌、抗霉菌、及抗癌药物标的之 潜力,已吸引科学家对其投入超过半个世纪以上时间之研究。 本实验室利用基因遗传工程技术以及产物分離和鉴定等策略对酵母菌之氧化鲨烯环 化酵素所催化之环化重组反应之结构-功能-作用机制关系有初步之了解。我们在过去以 來,已确认數个对环化或重组过程中扮演重要角色之胺基酸,并由氧化鲨烯环化酵素之 变种中分離數个在环化或重组过程中被中断之中间产物。这些结果支持酵素之结构-功 能-作用机制关系以及提供作为未來药物应用之基础。虽然截至目前所获得之结果甚为 樂观,但若能对蛋白质之可塑性及所产生产物专一性或多样性之过程有进一步之了解, 将可对将來将此類在氧化鲨烯环化过程之中间产物应用于降胆固醇或抗霉菌药物之开 发有所帮助。另外若在所产生之中间产物,利用三萜類生合成之修饰酵素进行官能基或 衍生物之修饰,将可增加其结构之多样性以及帮助其生理活性。 为了达到上述之目标,我们将继续探讨酵母菌之氧化鲨烯环化酵素之结构-功能-作用 机制关系,研究蛋白质之混杂性,及设计具新功能之酵素。同时我们也将选殖及表现三 萜類生合成途径之修饰酵素如氢氧化酵素、醣基转移酵素、及醯基转移酵素。经由酵素 修饰作用将可增加所获得三萜類中间产物之结构多样性。所修饰之三萜類中间产物将用 于抗菌及抗癌药物之活性测试。 因此,本计画在未來三年之工作目标将进行以下數项之工作: (1) 针对氧化鲨烯环化酵素之可能进行分歧演化之区域进行蛋白质工程研究。 A. 氧化鲨烯环化酵素之分歧演化之区域之生物资讯分析。 B. 氧化鲨烯环化酵素之分歧演化之区域之蛋白质工程。 C. 利用2,3:22,23-双氧化鲨烯做为可能之受质以增加其官能基之可行性。 D. 利用GC-MS 及LC-MS 分析所产生之截短或神奇之产物。 (2) 分子选殖及蛋白质表现三萜類生合成途径之修饰酵素及裁制修饰截短或神奇之 产物。 A. 分子选殖及蛋白质表现三萜類氢氧化酵素及裁制修饰截短或神奇之产物。 B. 分子选殖及蛋白质表现三萜類醣基转移酵素及裁制修饰截短或神奇之产物。 C. 分子选殖及蛋白质表现三萜類醯基转移酵素及裁制修饰截短或神奇之产物。 (3) 三萜類新产物之抗肿瘤及抗细菌和抗霉菌生理活性分析。 A. 细胞培养准备以进行药物效应分析。 B. 评估所产生之截短或神奇之产物或其裁制修饰物之细胞毒性。 C. 评估所产生之截短或神奇之产物或其裁制修饰物对细胞增生及程式性凋亡之 影响。 D. 评估所产生之截短或神奇之产物或其裁制修饰物于程式性凋亡过程之形态变 化。 E. 评估所产生之截短或神奇之产物或其裁制修饰物于DNA 断裂之分析。 F. 评估所产生之截短或神奇之产物或其裁制修饰物于抗菌活性之分析。 G. 评估所产生之截短或神奇之产物或其裁制修饰物于抗霉菌活性之分析。 本计画之执行,对于氧化鲨烯环化酵素之结构-功能-作用机制之关系将可有进一步之 了解,并可能产生具新功能或能产生新产物之新酵素,增加所产生截短或神奇之产物或 其裁制修饰物之结构多样性,以及提供作为未來获得具氧化鲨烯环化酵素抑制活性之抑 制剂及其作为抗细菌、抗霉菌、及抗癌药物之应用。 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. |
官方说明文件#: | NSC99-2113-M009-008-MY3 |
URI: | http://hdl.handle.net/11536/98311 https://www.grb.gov.tw/search/planDetail?id=2391851&docId=380447 |
显示于类别: | Research Plans |