標題: 重要抗生素C-甲基化的生合成分子機理研究與在新穎抗癌藥物的研發應用
Molecular Mechanism and Biosynthetic Study of Antibiotic C-Methylations and Their Applications in Research and Developments of Novel Anticancer Agents
作者: 邱顯泰
Chiu Hsien-Tai
國立交通大學生物科技學系(所)
關鍵字: 生物合成;C-甲基化;酵素學;抗生素;二磷酸核?醣;含醣??化合物;藥物研發;Biosynthesis;C-Methylation;Enzymology;Antibiotics;NDP-sugar;IndolocarbazoleGlycosides;Drug Development
公開日期: 2009
摘要: 生物活性分子的甲基化是自然界的一種重要機制,其參與細胞各項活性機能並操控生物分子 的生物活性,譬如細胞分化、基因表現的調控、後轉譯修飾、tRNA 的成熟、抗藥機制、及天然 物的生物合成等。在無以數計的具有重要生物活性的天然物中,常發現具有結構多樣性的醣分子 成份。研究顯示,該醣分子為其生物活性所必須,且其醣基的化學結構,可決定該天然物的效能 與專一性。值得注意的是,這些醣分子的碳骨架上常帶有被C-甲基轉移酵素修飾的甲基,此C- 甲基化,使其醣分子更具有多樣性的分子構型,並調控其疏水性分布。因此,瞭解這些醣類C- 甲基轉移酵素的分子認知與機制,及其在天然物生物合成中扮演的角色,便成為一有趣且重要的 研究課題。為探討此課題,本研究即從四個具有抗癌、抗腫瘤與抗生素活性的含醣天然物生合成 途徑中,找得四種代表性類型(含三號碳與五號碳)的C-甲基轉移酵素,以進行其功能性表現、分 子機制與受質專一性的研究。 本研究從產生此四種天然物的微生物中,選殖、表現並純化這四種C-甲基轉移酵素,以二磷 酸核苷四號酮基六號去氧葡萄醣及其光學異構物,進行功能性表現與產物化學結構鑑定。而後, 並使用一群四號酮基醣受質類似物,作為探測酵素活性催化中心的立體結構的探針,進行一系列 酵素動力學與抑制學實驗,統整分析其動力學結果,以破解其受質分子認知與催化機制的原理, 並解析歸納不同C-甲基轉移酵素的演化差異與特性。另外,本研究將以定點突變的方式,配合 酵素動力學,鑑定出催化機制中扮演鹼基與重要功能的活性中心胺基酸。此外,研究中亦探討並 比較此四種酵素的生化特性,如金屬離子依賴性、酸碱值與酵素活性的關係等。 另一研究主題是,本研究將利用此四種不同C-甲基轉移酵素,以組合式化學的方式,催化合 成一群甲基化二磷酸核苷醣分子資料庫。此實驗可以四種C-甲基轉移酵素混合排列,進行各式 酮基醣的C-甲基化而成,並可再進一步搭配兩種立體催化化學不同的四號酮基去氧醣還原酵 素,或化學還原法,而得還原形式的各式甲基化二磷酸核苷醣分子。此醣分子資料庫可充份應用 於醣生物學,及天然物生合成與醣基化研究上。例如,本研究將應用此醣分子庫,以抗生素N- 醣基轉移酵素來轉移各式甲基化醣基,以修飾並合成一群含醣吲哚類抗癌抗腫瘤化合物,以作為 抗癌症藥物的研發與應用。此研究成果並將有助於解決現今日益嚴重的抗生素與抗癌藥物的抗藥 性及副作用問題。
Methylation of biologically active molecules has been an important mechanism of nature to control cellular processes and to monitor biological activity of biomolecules, including cell differentiation, gene expression regulation, posttranslational processes, tRNA maturation, antibiotic resistance and natural product biosynthesis. Many important bioactive natural product glycosides bear structurally diverse sugars indispensible for their biological potency and specificity. Notably, many of these sugar moieties were found to be C-methylated by various C-methyltransferases (C-MTases), thereby leading to control of structural conformation and global hydrophobic properties of the sugar moieties. Therefore, it would be an interesting and important task to study the biosynthetic origin and mechanism of the C-methylation in these biologically important glycosylated natural products, serving as antibiotics, antitumor and anticancer agents. In this study, we have chosen four valuable natural product glycosides of this kind, i.e. coumermycin A1, novobiocin, erythromycin and nogalamycin, to investigate the molecular mechanism and substrate specificity of the four C-MTases involved in their biosyntheses. To this aim, a series of TDP-4-keto-sugar analogs will be used as structural and mechanistic probes to explore the active-site cavities of the C-MTases. The study may be realized by extensive steady-state and inhibition kinetic experiments using the C-MTases cloned, expressed and purified from the drug-producing organisms. The resulting systematic kinetic information will be very useful to resolve the structure-activity relationship of the enzymes. The C-MTases will also be subjected to intensive biochemical characterization, e.g., metal ion requirements and pH-dependent activity profiles. In addition, site-directed mutagenesis will be conducted to identify the important active-site residues involved in the chemical catalysis of the C-MTases. Another interesting study is to utilize these four C-MTases as a synthetic tool for combinatorial biosyntheses of a variety of methylated nucleotide diphosphate sugars (NDP-sugars). The synthesis may be accomplished by catalytic actions of the C-MTases on the TDP-4-keto-sugar analogs, further coupled with NDP-sugar reductases or chemical reduction. The resulting methylated NDP-sugar library can be valuable for various glycosylation events in glycobiology and natural product biosynthesis. In this study, the methylated analog pool of NDP-sugars will be utilized for molecular engineering of the glycosylated indolocarbazole family of antitumor antibiotics by N-glycosyltransferases, which may decorate the indolocarbazole aglycones with various methylated sugar molecules. The indolocarbazole glycosides generated in this study may thus serve as a valuable source of drug leads for anticancer drug developments and therapeutic applications.
官方說明文件#: NSC98-2113-M009-003
URI: http://hdl.handle.net/11536/101684
https://www.grb.gov.tw/search/planDetail?id=1880583&docId=310532
顯示於類別:研究計畫