探討去氧鬼臼毒素合酶催化氧化合環反應之結構研究

Abstract

鬼臼毒素 (podophyllotoxin) 是一種非生物鹼類、木脂素類毒素，其衍生化合物是臨床上普遍使用的抗癌或抗病毒藥物，分別主要抑制細胞中的第二型拓樸異構酶 (topoisomerase type II) 活性以及細胞骨架微管 (microtubule) 的聚合而造成療效。鬼臼毒素主要的天然來源為桃兒七和美洲鬼臼草，這兩株植物相對其他自然資源含有較大量的活性成分，在過去幾年因應臨床上的大量需求多為人採伐，加上棲地遭受危害，成為了瀕危物種。因此，近年來開啟了許多對於鬼臼毒素生合成路徑的研究，希望透過基因工程以及分子生物學技術，將原生物體內之代謝途徑移植至模式生物中，代替產生所需化學物質。去氧鬼臼毒素合酶 (deoxypodophyllotoxin synthase, DPS) 是參與在木脂素生合成途徑的重要蛋白，完成芳基四氫萘(aryltetralin) 主結構。DPS 屬於為一非血基質鐵 (non-heme iron) /α-酮戊二酸 (α-ketoglutarate) 依賴型雙氧化酶蛋白家族，家族成員皆擁有類似的活性中心以及催化機制。其活性中心的二價鐵離子以六配位方式，由兩個組胺酸 (Histidine) 的氮與另一天門冬胺酸 (Aspartic acid) 或麩胺酸 (Glutamic acid) 之羧基所構成的 2-His-1-carboxylate motif 螯合，其餘三個配位則由水分子所佔據。此外，整個活性中心被八個反向平行β-摺板所形成穩定的double-stranded β-helix 結構包覆。此蛋白家族的共通催化機制一開始是氧氣活化形成ferryl-oxo 單元，此單元具有強氧化力，不同的酵素針對特定的受質便能夠驅使多樣化的受質攻擊。DPS 催化具有立體專一性的氧化合環作用，將受質 (–)-yatein 轉化成 (–)-deoxypodophyllotoxin。先前報導曾提出可能的催化機制，DPS 在受質的C 環七號碳上進行奪氫反應，產生碳陽離子或受質自由基的中間產物，並促使去質子化生成碳碳鍵 (C-C) 合環。然而，目前尚未解出DPS 蛋白結構，因而缺乏結構方面的證據來支持上述的假設。因此，本篇論文主要以蛋白結構的角度切入，探討DPS 如何誘導C 環上二號碳與七號碳之間的鍵結生成與合環，包括其催化機制以及如何控制產物的掌性。目前已成功表現帶有組胺酸標籤 (His tag) 的可溶性DPS 並且透過固定化金屬離子親和性層析、陰離子交換層析、膠體過濾層析成功得到高純度的DPS。透過氣相擴散法進行晶體培養，目前分別成功獲得輔因子結合態 (DPS•Fe•2-OG) 以及受質結合態(DPS•Fe•succinate•DMOY) 的晶體，目前收集到最好的數據之解析度為2.09 Å。為了解決相位角問題，我們則成功依循前面純化方式培養出已含有硒化甲硫胺酸 (Selenomethionine) 之DPS 晶體，進行多波長非尋常散射法 (multi-wavelength anomalous diffraction)。然而，由於晶體小且數據解析度不夠，晶體仍待再優化。因此，培養出能進行相位角解析的晶體是目前想要解出DPS 結構的當務之急。未來希望透過與不同受質類似物或產物類似物的晶體結構，解析出DPS 催化氧化合環反應的完整過程。

Derivatives of podophyllotoxin, a non-alkaloid lignan, are clinically active anticancer and antiviral agents whose mechanisms of action involve direct targeting of eukaryotic type II topoisomerases and destabilizing microtubules. Despite being highly demanded for its medical usage, the production capacity of podophyllotoxin is limited by the availability of its nature sources Sinopodophyllum hexandrum and Podophyllum. A better understanding of the biosynthetic pathway of podophyllotoxin is expected to benefit the development of an alternative procedure for producing this important compound by metabolic engineering. Deoxypodophyllotoxin synthase (DPS) is one of the key enzymes involved in the podophyllotoxin biosynthesis. DPS belongs to the Fe(II)/2OG-dependent oxygenase superfamily, which features the presence of a 2-His-1-carboxylate motif for coordinating Fe(II) and a distorted double-stranded β-helix (DSBH) fold composed of 8 antiparallel β-strands. The common catalytic cycle of this family starts from oxygen activation to generate Fe(IV)-oxo active center, and this highly reactive Fe(IV)-oxo species is capable of triggering a variety of oxidative transformations on different substrates. DPS catalyzes stereoselective oxidative ring closure during the conversion of (–)-yatein to (–)-deoxypodophyllotoxin. According to a proposed mechanism, DPS initiates benzylic hydrogen atom abstraction on the C’7 of its substrate and generates a carbocation or substrate radical, which serves as an intermediate tofacilitate deprotonation and subsequent Carbon-Carbon (C-C) bond formation. Nevertheless, the structural basis governing DPS function has remained to be explored. Therefore, the main goal of my thesis research is to elucidate how DPS mediates the oxidative cyclization via C-C bond formation between C2 and C7. To this end, we have produced His-tagged DPS and successfully obtained highly purified protein through immobilized metal affinity chromatography, anion exchange chromatography and size-exclusion chromatography. Using the vapor diffusion method, both the 2-OG-bound and substrate-bound DPS have been crystallized, and a complete diffraction data set has been collected to 2.09 Å resolution. To solve the phase problem, we have prepared selenomethionine-derivatized DPS crystals to allow the application of selenium-based anomalous diffraction (Se-MAD) phasing method, however, further improvement is needed to get high-quality crystals. We have employed various crystallization strategies, such as micro-seeding, but with no crystal formation yet. Therefore, to establish a robust crystallization scheme and produce high-quality Se-DPS crystals are the top priority for structural studies on DPS. Moreover, crystallization of DPS complexed with other chemical combinations that represent distinct stages of the catalytic cycle will also be performed in the future.