Functional cloning of an Arabidopsis thaliana cDNA encoding cycloeucalenol cycloisomerase
Functional cloning of an Arabidopsis thaliana cDNA encoding cycloeucalenol cycloisomerase
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DOI：
10.1074/jbc.275.18.13394
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发表时间：
2000-05-05
影响因子：
4.8
通讯作者：
Matsuda, SPT
中科院分区：
文献类型：
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作者：
Lovato, MA;Hart, EA;Matsuda, SPT
植物和某些原生生物利用环桉烯醇环异构酶（EC 5.5.1.9）将五环环丙基甾醇转化为常规四环甾醇。我们利用一种新的互补策略克隆了环桉烯醇环异构酶cDNA。在酵母羊角甾醇合成酶突变体中表达拟南芥环蒿烯醇合成酶cDNA，提供了一个可以与异构酶遗传互补的甾醇营养不良菌。我们用拟南芥酵母表达文库对该酵母菌进行转化，并选择甾醇原生营养物，获得了一个积累生物合成麦角甾醇的菌株。这种新表型是由拟南芥cDNA赋予的，该cDNA可能编码一个36 kda的蛋白。我们在大肠杆菌中表达了该cDNA (CP11)，并通过气相色谱-质谱分析表明，该菌株的提取物在体外可将环桉烯醇异构化为烟叶油醇。该cDNA将有助于获得异源表达蛋白，用于催化研究和阐明环丙基甾醇在体内的作用。
Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase, We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein. We expressed this cDNA (CP11) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.