特殊去氧醣胺基轉移酵素的分子機制研究與其在胺基醣藥物的組合式生合成應用

Abstract

胺基化對生物體內的有機分子的代謝、結構多樣化與功能化上扮演重要的角色。尤其，很多具 生物活性的含醣次級代謝物因具有特殊胺基醣而具有重要生物活性，如抗生素、抗腫瘤、抗癌、甚 至抑制害蟲的活性。在自然界中，這些胺基醣化合物皆需藉由二磷酸核苷醣胺基轉移酵素而得以用 立體與位向專一性的方式，將胺基轉移至各式的醣分子結構上。這類酵素如何演化，而在辨識並包 容各式醣類結構下轉移胺基，已儼然成為有趣且重要的研究課題。經由我們許多初步的研究成果顯 示，此類胺基轉移酵素對醣分子受質具有驚人的受質不專一性，這結果對藉以製作各式胺基醣，來 增加胺基醣藥物的結構多樣性，具重要影響與意義。 鑒於上述的前景與重要性，本計畫將以三號與四號胺基轉移酵素為研究對象，徹底解析在此類 酵素活化中心的結構與活性關係。另外，由於現今對此特別族群的受質專一性與反應機制的所知有 限，本計畫亦特別選擇三種重要天然藥物的去氧醣胺基轉移酵素來探討上述課題。主要策略是，以 化學或生物合成一群二磷酸胸腺嘧啶三號及四號酮基醣類化合物，來作為探測酵素活性中心結構的 分子探針，並以一連串有系統的動力學(包含抑制動力學)實驗，來鑑定酵素的受質專一性，及立體 與位向專一性。同時，並利用蛋白質定點突變法，配合酵素動力學，來搜尋參與化學催化和受質辨 識的重要活性胺基酸的功能與角色，一方面亦可藉由酵素突變種來獲得更佳的受質包容度。這些實 驗結果，將得以藉由轉胺作用來合成一群各式的二磷酸核苷胺基醣，並進一步用來胺基醣化吲哚醣 苷，而得各種具有藥物潛力的吲哚胺基醣衍生物，作為疾病治療之應用。

Amination has been an important approach adopted by Nature to diversify organic molecules with specific functions indispensable for metabolism. In particular, many bioactive secondary metabolite glycosides possess aminosugars critical for their reported biological activities and potency, including antibiotic, antitumor, anticancer and insecticidal agents. Biosyntheses of the aminoglycosides in nature require catalytic actions of NDP-sugar aminotransferases responsible for transferring the amino group onto their carbohydrate moieties in specific stereochemistry and regioselectivity. How such enzymes evolve to possess the ability to accommodate their substrates and to direct the transfer has clearly become an important and interesting issue to be investigated. In addition, our preliminary studies have shown NDP-sugar aminotransferases exhibit remarkable substrate flexibility towards the carbohydrate moiety, which can be very useful in generating structurally diverse aminosugars to expand the glycosylation pattern of bioactive aimoglycosides. In light of this promise, this proposal aims to study the structure-activity relationship taking place in the active sites of the enzymes, the 3-aminotransferases and 4-aminotransferases in particular. Due to the lack in detailed information about molecular mechanism in substrate specificity and chemical catalysis for the enzymes, we have targeted these two types of enzyme with EryCI, OleN2 and SpnR to address the questions. The main approach we make is to probe the enzyme active site with a variety of TDP-3-keto-sugars and TDP-4-keto-sugars which will be synthesized in this study by both chemical and biosynthetic methods. The characterization of substrate specificity will be made on these substrate analogs by a series of kinetic experiments including inhibition kinetics. In addition, site-directed mutagenesis will be attempted to investigate the functional roles of some potential amino acids involved in chemical catalysis and substrate binding, as well as to engineer the enzymes for broader substrate flexibility. Consequently, the resulting products from transamination will give a pool of various TDP-aminosugars that can be utilized by an indolocarbazole glycosyltransferase to make a group of indolocarbazole aminoglycosides for practical applications of this study in drug discovery and development.