解析牛樟芝的雙功能萜烯環化酶/磷酸酶以及相關之基因體分析

Abstract

牛樟芝(Antrodia cinnamomea)是一種臺灣傳統使用的中藥。在本實驗室先前的研究中，我們已經利用異源表達以及前驅物生物轉化的實驗闡明了牛樟芝活性化合物antrocin的生物合成途徑。在本篇研究中，我們使用生化方法以進一步了解參與antrocin生物合成途徑的催化酶功能。 在antrocin的生物合成途徑中，為了瞭解細胞色素P450氧化酶−AncB和AncD的功能，我們分離其微粒體萃取物，以測試P450氧化酶對於albicanol的氧化能力。另外，有兩個haloacid dehalogenase-like (HAD-like)的催化酶−AncA和AncC。AncC已知是一類新型的萜烯環化酶，具有兩種催化特性，(i)法尼基焦磷酸farnesyl pyrophosphate (FPP)的環化，生成albicanol pyrophosphate (APP)，以及(ii) APP的脫磷酸作用，形成albicanol。AncA與AncC具有高度的序列相同性和相似性，但AncA僅具有去磷酸化的活性。我們將AncA、AncC與常見的HAD磷酸酶的序列作比對，發現AncA和AncC的N端區域與常見的HAD磷酸酶可以對齊並具高序列相似性。因此，我們純化了僅有N端區域的AncC，並確認它亦具有磷酸酶的活性。對於C端區域，我們對AncC做了18個點突變，藉由GC-MS分析來探討每個突變點所帶來的影響，發現了其中六個位點會降低albicanol的生產。因此，我們將這六個位點在AncA中重建，得到的AncA突變在酵母菌表達後顯示具有微弱的環化活性。 此外，我們也對子囊菌真菌(Ascomycete)進行了基因採礦分析，發現其中具有許多潛在的萜烯環化酶。我們建立萜烯環化酶演化樹，並在不同分枝中選擇了四個催化酶進行表達與功能分析，以了解其催化特性。我們的研究探討了一個新家族的HAD萜烯環化酶的催化機制，並探索其同源酶的催化功能。

Antrodia cinnamomea is a folk medicine traditionally used in Taiwan. In our previous study, we have elucidated the biosynthetic pathway of antrocin, one of the cytotoxic constituents from A. cinnamomea, with heterologous expression and in vivo feeding experiments. In this study, we performed biochemical methods to characterize the enzymes involved in the biosynthetic pathway of antrocin. In antrocin biosynthesis, to characterize the function of cytochrome P450 monooxygenases, AncB and AncD, we isolated the microsome extracts to test the oxidization property on albicanol. Two haloacid dehalogenase-like (HAD-like) enzymes, AncA and AncC, also participate in the antrocin pathway. AncC has been known as a new class of terpene cyclase that showed two catalytic properties, (i) cyclization of farnesyl pyrophosphate to give albicanol pyrophosphate (APP) and (ii) dephosphorylation of APP to form albicanol. AncA showed high sequence identity and similarity to AncC, but it only showed dephosphorylation activity. Sequence alignment of AncA, AncC and classical HAD-phosphatases showed that the N-terminal domain of AncA and AncC aligned well with the classical HAD-phosphatase. We purified the N-terminal domain of AncC and confirmed that it showed phosphatase activity. For the C-terminal domain, we have done eighteen point mutations on AncC and then analyzed the function of each mutant by the combination of in vivo experiments and GC-MS analysis. Six motifs were found to involve in the production of albicanol. These six motifs were reconstituted in AncA, and the mutated AncA showed weak cyclization activity. Furthermore, we performed gene mining analysis in Ascomycete fungi and found that many of them contain the homologs of terpene cyclases. Four enzyme candidates from the phylogenetic analysis have been selected and cloned to characterize their catalytic properties. Our study characterized the catalytic mechanism of the new family of HAD-like terpene cyclase and explored the function of their homologs in Ascomycete fungi.