Nes W R, Dhanuka I C
Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104.
J Biol Chem. 1988 Aug 25;263(24):11844-50.
Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.
在酿酒酵母的GL7突变体中证实了麦角固醇的合成。这种甾醇营养缺陷型一直被认为缺乏合成甾醇的能力，原因是既没有2,3-氧化角鲨烯环化酶，又因血红素缺乏而消除了C-14脱甲基化所需的细胞色素P-450。然而，当培养基中的甾醇为5α-胆甾烷-3β-醇、5α-胆甾-8(14)-烯-3β-醇或24β-甲基-5α-胆甾-8(14)-烯-3β-醇时，发现甾醇合成得以进行，产生1-3 fg/细胞的麦角固醇（24β-甲基胆甾-5,7,22E-三烯-3β-醇）。通过质谱、气相和高效液相色谱、紫外光谱以及来自[3H]乙酸盐的放射性标记鉴定了麦角固醇。除了一些源自5α-胆甾烷-3β-醇的胆甾-5-烯-3β-醇（胆固醇）外，甾烷醇和两种8(14)-甾烯醇没有明显代谢，这证实了不存在将双键从C-8(14)迁移到C-7的异构酶。当培养基中的甾醇在C-5处有双键（如胆固醇的情况）或可代谢为具有这种双键的甾醇时，观察到麦角固醇合成急剧减少至不超过0.06 fg/细胞。因此，5α-胆甾-8(9)-烯-3β-醇和5α-胆甾-7-烯-3β-醇（羊毛甾醇）都转化为胆甾-5,7-二烯-3β-醇（7-脱氢胆固醇），而后一种二烯醇的存在降低了麦角固醇的水平。对我们观察结果最有吸引力的可能解释是假设甾醇合成存在两个遗传区室，其中一个受到了两个突变的影响，而另一个没有。尽管发生了突变，但仍有能力合成少量可用于调节细胞周期的麦角固醇，这也可以解释为什么这种突变体能以胆固醇（在质膜中起主要作用）作为唯一的外源甾醇进行有氧生长。
