Lecain E, Chenivesse X, Spagnoli R, Pompon D
Centre de Génétique Moléculaire du CNRS, Laboratoire propre associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
J Biol Chem. 1996 May 3;271(18):10866-73. doi: 10.1074/jbc.271.18.10866.
Reduction of the delta 7 double bond of sterols, a key biosynthetic step in higher eukaryotes, is lacking in lower eukaryotes like the yeast Saccharomyces cerevisiae, leading to terminal sterols with a delta 5,7-conjugated diene structure. Genes encoding two sterol reductases involved, respectively, in the reduction of sterol delta 14 and delta 24(28) double bonds have been cloned to date, but no sequence information was available on the enzyme responsible for delta 7-bond reduction. This study presents the cloning of the NADPH-sterol delta 7-reductase (delta 7-red) from Arabidopsis thaliana, based on a metabolic interference approach in yeast. The principle is the functional expression of a plant cDNA library in the yeast strain FY1679-28C tolerant to sterol modifications and the selection of clones resistant to the polyene fungicide nystatin. The toxicity of this compound is dependent on the presence of delta 5,7-unsaturated sterols in the yeast plasma membrane. One clone out of 10(5) transformants exhibits a cDNA-dependent alteration of cell sterol composition. The 1290-base pair cDNA open reading frame was isolated and sequenced. The corresponding protein presents a significant sequence similarity with yeast delta 14- and delta 24(28)-reductases and with human lamin B receptor. The coding sequence was extracted by polymerase chain reaction and inserted into a galactose-inducible yeast expression vector to optimize expression. Analysis using transformed wild type yeast or sterol altered mutants, indicated that delta 5,7-ergosta- and cholesta-sterols are efficiently reduced in vivo, regardless of the structural variations on the side chain. No reductase activity was observed toward the delta 14 or the delta 5 positions of sterols. In vivo extensive delta 7-reduction of the free and esterified pools of sterols was observed upon induction of the enzyme. Ergosterol present before induction was reduced into ergosta-5,22-dieneol, whereas ergosta-5-eneol is the new end product of sterol neosynthesis, indicating that the yeast delta 22 desaturase may be no longer active on C-7-saturated sterols. In vitro tests indicated that delta 7-reductase activity is preferentially associated with the endoplasmic reticulum membrane and confirmed the previous finding that NADPH is the reducing agent.
甾醇δ7双键的还原是高等真核生物生物合成中的关键步骤，而在像酿酒酵母这样的低等真核生物中则不存在这种还原过程，这导致其末端甾醇具有δ5,7 -共轭二烯结构。迄今为止，分别编码参与甾醇δ14和δ24(28)双键还原的两种甾醇还原酶的基因已被克隆，但负责δ7键还原的酶的序列信息尚无报道。本研究基于酵母中的代谢干扰方法，报道了从拟南芥中克隆NADPH -甾醇δ7 -还原酶（δ7 - red）。其原理是在对甾醇修饰具有耐受性的酵母菌株FY1679 - 28C中功能性表达植物cDNA文库，并筛选对多烯类杀菌剂制霉菌素具有抗性的克隆。该化合物的毒性取决于酵母质膜中δ5,7 -不饱和甾醇的存在。在10^5个转化体中，有一个克隆表现出依赖于cDNA的细胞甾醇组成改变。分离并测序了1290个碱基对的cDNA开放阅读框。相应的蛋白质与酵母δ14 -和δ24(28) -还原酶以及人类核纤层蛋白B受体具有显著的序列相似性。通过聚合酶链反应提取编码序列，并将其插入半乳糖诱导型酵母表达载体中以优化表达。使用转化的野生型酵母或甾醇改变的突变体进行分析表明，无论侧链上的结构变化如何，δ5,7 -麦角甾醇和胆甾醇在体内都能被有效还原。未观察到对甾醇δ14或δ5位置的还原酶活性。在诱导该酶后，在体内观察到甾醇的游离和酯化池均发生了广泛的δ7 -还原。诱导前存在的麦角甾醇被还原为麦角甾 - 5,22 -二烯醇，而麦角甾 - 5 -烯醇是甾醇新合成的新终产物，这表明酵母δ22去饱和酶可能对C - 7饱和甾醇不再具有活性。体外试验表明，δ7 -还原酶活性优先与内质网膜相关，并证实了之前NADPH是还原剂的发现。
