牛樟芝中之倍半萜烯合成酶AcCOP4結構與功能探討

Structural and Functional Characterization of a Sesquiterpene Synthase AcCOP4 from Antrodia cinnamomea

江柏辰(Bo-Chen Jiang)

指導教授 ： 徐駿森

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202104403

摘要

萜類化合物（terpenoids）占了近1/3已知的天然化合物組成，是由各類型的萜烯合成酶（terpene synthase）進行延長與環化反應而產生。其中倍半萜烯合成酶（sesquiterpene synthase）可使用FPP作為受質，生產多種具揮發性質的倍半萜類化合物。而牛樟芝（Antrodia cinnamomea）是臺灣本土的特有真菌，被用做中藥材使用許久，被發現含有不少包含萜類化合物的活性成分。從先前轉錄體研究發現有18個可能為萜烯合成酶的基因，經過序列與親緣關係的分析以及蛋白質表現測試，選定其中AcCOP4（ACg002067）進行後續的蛋白質特性分析、活性測定以及結構解析等實驗。以GC-MS分析AcCOP4以FPP為受質的反應，發現至少有五種產物生成，並以germacrene D與cubebol為主要，顯示了此酵素的催化多樣性。利用圓二色光譜（CD）得知蛋白在pH 5.0~pH 10.0間二級結構的特徵峰較為明顯，並可判斷其二級結構以α-helix為主。而微量差示掃描螢光（nanoDSF）分析AcCOP4酵素，發現於輔因子Mg2+以及反應產物pyrophosphate (PPi)的同時存在，相比於PPi或Mg2+單獨存在時，蛋白質Tm值有微幅的變化。利用分析級膠體過濾層析法（analytical gel filtration chromatography），推算出AcCOP4以單體的形式存在。經過晶體篩選、培養與繞射數據收集後，利用分子置換法（molecular replacement）解析出了AcCOP4與AcCOP4-Mg2+3-PPi複合體的結構，為一個以α-helices為主的典型Class Ⅰ萜烯合成酶結構。酵素中央空腔內的Asp87、Glu157、Arg176、Asn222、Ser226、Glu230、Arg311與Tyr312，可與PPi產生氫鍵或與Mg2+有配位鍵的形成，可能造成酵素構型改變。AcCOP4的晶體結構為首個源自牛樟芝的萜烯合成酶結構，我們在此利用解析出之結構，對其催化特性與功能進行分析與探討。

關鍵字

牛樟芝 ； 萜類 ； 萜烯合成酶 ； 萜烯環化酶 ； 催化多樣性 ； 晶體結構

並列摘要

The terpenoid accounts for the majority of natural products, which are produced by various types of terpene synthase. The sesquiterpene synthase would utilize the FPP as the substrate to produce numerous volatile sesquiterpenoid products. The Antrodia cinnamomea is an indigenous species that have activity ingredient and used as traditional medicine. In previous research, A. cinnamomea contains at least 18 putative terpene synthases. After sequence analysis, phylogenetic analysis, and expression test of putative terpene synthases, we selected AcCOP4(ACg002067) as the target protein. The GC-MS analysis shows that AcCop4 produces five different products, and germacrene D and cubebol are two major products. This phenomenon shows the catalytic promiscuity of AcCOP4. The CD results indicate the main secondary structure of AcCOP4 is composed of α-helical. The nanoDSF analysis shows Tm have subtle changes when Mg2+and PPi are coexisting. The results of analytical gel filtration show it exists as a monomer in solution. After crystal screening, growth, and diffraction data collecting, we use the molecular replacement deciphering the AcCOP4 crystal structure. The structure of AcCop4 and AcCOP4-Mg2+3-PPi reveals enzyme adopts the typical α-helical class I terpene synthase fold. The Mg2+ and PPi can be coordinated or create hydrogen bonds with Asp87, Glu57, Arg176, Asn222, Ser226, Glu230, Arg311, and Tyr312 residues in the enzyme cavity. These interactions might cause AcCOP4 conformational change. Up to now, AcCOP4 is the first structure of terpene synthase from A.cinnamomea. We use the deciphered crystal structures for analyzing and discussing protein function.

並列關鍵字

Antrodia cinnamomea ； Terpenoids ； Terpene synthase ； Terpene cyclase ； Catalytic promiscuity ； Crystal structure

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