Functional cloning of an <i>Arabidopsis</i> <i>thaliana</i> cDNA encoding cycloeucalenol cycloisomerase

作     者：Lovato, MA Hart, EA Segura, MJR Giner, JL Matsuda, SPT 

作者机构：Rice Univ Dept Chem Houston TX 77005 USA Rice Univ Dept Biochem & Cell Biol Houston TX 77005 USA SUNY Dept Chem Syracuse NY 13210 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：2000年第275卷第18期

页      面：13394-13397页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NIAID NIH HHS [AI41598] Funding Source: Medline NIGMS NIH HHS [T32 GM08362] Funding Source: Medline 

主　　题：氨基酸序列 拟南芥属/遗传学 拟南芥蛋白质类/遗传学 碱基序列 克隆 分子 DNA 互补/遗传学 DNA 互补/分离和提纯 大肠杆菌 分子内裂合酶类/遗传学 异构酶类/遗传学 分子序列数据 

摘      要：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.