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首页> 美国卫生研究院文献>Proceedings of the National Academy of Sciences of the United States of America >Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis
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Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

机译：酿酒酵母ERG26基因的编码该基因编码参与固醇生物合成的C-3固醇脱氢酶（C-4脱羧酶）

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All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.
机译：已克隆了酿酒酵母中麦角固醇生物合成途径中涉及的除两个基因外的所有基因，并描述了其相应的突变体。其余的基因编码C-3固醇脱氢酶（C-4脱羧酶）和3-酮固醇还原酶，并与C-4固醇甲基氧化酶（ERG25）共同催化在固醇C-处两个甲基的顺序去除4位置。负责将胆固醇转化为其3-酮衍生物的诺卡氏菌NAD（P）依赖性胆固醇脱氢酶的蛋白质序列显示与329-aa酿酒酵母ORF YGL001c有30％的相似性，表明YGL001c在固醇脱羧中的可能作用。 YGL001c ORF的破坏是在一个二倍体菌株中进行的，并且将分离物在厌氧生长条件下接种到添加了固醇的培养基上。含有YGL001c干扰的分离子在转移到新鲜的，添加了甾醇的培养基后不可行。但是，一个隔离子能够生长，遗传分析表明它含有hem3突变。 YGL001c（ERG26）破坏在麦角固醇存在下生长的1Δhem菌株中也是可行的。在补充了麦角固醇的培养基中，也可以将erg26突变引入erg1（角鲨烯环氧酶）菌株。我们证明了在各种固醇和血红素补充的培养基上生长的erg26突变体会积累未酯化的羧酸固醇，例如4β，14α-二甲基-4α-羧基-cholesta-8,24-dien-3β-ol和4β-甲基-4α-羧基-cholesta-8,24-dien-3β-ol，是C-3甾醇脱氢酶的预测底物。这些固醇分子在血红素适应性erg26菌株中的积累导致有毒氧化的固醇中间体的积累，即使在存在外源添加的固醇的情况下，它们也阻止了生长。

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