標題: 諾加黴素之碳-甲基轉移酵素的定點突變研究
Site-Directed Mutagenesis Studies of Nogalamycin C-methyltransferase
作者: 翁允軒
邱顯泰
分子醫學與生物工程研究所
關鍵字: 諾加黴素;碳-甲基轉移酵素;去氧醣;定點突變;含醣天然物;nogalamycin;C-methyltransferase;deoxysugar;site-directed mutagenesis;natural product glycoside
公開日期: 2010
摘要: 天然物富含各種生物活性,如抗生素、抗癌藥劑、降血酯、免疫抑制與麻醉等,其中一類具有醣基修飾,稱為含醣天然物。含醣天然物的醣基與其生物活性相關,修改醣基的種類或數目常導致其活性改變或喪失,而甲基化的修飾更可影響醣基的極性與立體結構,具有特殊的意義。 本論文研究諾加黴素之醣類生合成基因中的碳-甲基轉移酵素(SnogG2),探討其催化活性與重要胺基酸間的關係。針對四個具重要功能之保留胺基酸進行定點突變,建構出四個突變株之蛋白質表現質體,分別為Y223F、E225A、H226A及N387I,並經由異體表達與蛋白質純化,得到突變株酵素。接著以二磷酸胸苷-4-酮基-6-去氧-D-葡萄醣(dTDP-4-keto-6-deoxy-D-glucose,KDG)為受質,以硫-腺嘌呤核苷甲硫胺酸(S-adenosylmethionine,SAM)為輔酶進行酵素活性分析,再利用HPLC分析各突變株酵素之產物變化,並與野生株比較,以探討各胺基酸的重要性。 由本論文發現,不同的突變株酵素之活性與野生株差異極大,產物生成之比例亦不相同,若進一步再針對此酵素的其他重要胺基酸進行研究,深入了解酵素活性、催化機制與胺基酸序列間之關係,以期利用這些資訊作為蛋白質工程之依據,亦可產生各種不同甲基醣產物,應用於新穎含醣天然藥物之研發。
Deoxysugars are important structural components for exhibiting the biological activities of natural product antibiotics as well as numerous anticancer, antibacterial, and antifungal agents of diverse biosynthetic pathways. These deoxysugars play crucial roles in many natural products and the removal of deoxysugars often results in the loss or reduction of biological activity. Nogalamycin, a natural product produced by Streptomyces nogalater, has been studied as an antibiotic antitumor agent. Its unique structure contains a planar aglycon moiety and two sugar residues that bind to target DNA. One of the sugar residues is nogalose, an unusual methyl sugar that may participate as a DNA binder. Importantly, the C-3 methylation is catalyzed by C-methyltransferase(SnogG2) to biosynthesize the branched sugar. The methylation of deoxysugar would confer significant changes to the physiochemical properties by altering their solubility, reactivity and interaction with cellular targets. In this study, we identified and studied several amino acids possibly involved in substrate binding and catalytic mechanism by site-directed mutagenesis. The results showed interesting characteristics of mutant enzymes different from that of the wild-type. The observation has thus moved a step forward towards altering biological activities and potency for future molecular engineering of glycosylated natural products.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT079829514
http://hdl.handle.net/11536/47748
Appears in Collections:Thesis