Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium (.) . It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from , which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an αβ architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

中文翻译：

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涉及自由基黄素 delta-24 去饱和酶和钼依赖性 C26 羟化酶的细菌豆甾醇降解

甾醇是普遍存在的膜成分，由于其水不溶性和化学惰性而在很大程度上持续存在于环境中。最近，在胆固醇生长的反硝化细菌中发现了不依赖加氧酶的甾醇降解途径(.)。它通过四步 ATP 依赖性酶级联中的磷酸化中间体，实现类异戊二烯侧链未激活的初级 C26 羟基化为烯丙醇。然而，该途径与广泛分布的类异戊二烯侧链 C22 处含有双键的类固醇（例如植物甾醇豆甾醇）的降解不相容。在这里，我们从 中富集了一种原型 delta-24 去饱和酶，它催化中间体 stigmast-1,4-diene-3-one 到共轭 (22,24)-diene 的电子受体依赖性氧化。我们提出了 440 kDa 酶的 αβ 结构，每个亚基将黄素单核苷酸辅因子与组氨酰残基共价结合。分离时，两种黄素均以红色半醌自由基形式存在，可被柱头-1,4-二烯-3-酮还原，但即使用强氧化剂也不能氧化。我们提出了一种涉及烯丙基自由基中间体的机制，其中两个黄素半醌各自从底物中提取一个氢原子。形成的缀合 delta-22,24 部分允许随后通过异源产生的钼依赖性类固醇 C26 脱氢酶 2 与水对末端 C26 进行羟基化。总之，为 delta-22 类固醇阐明的途径实现了不依赖于氧的羟基化类异戊二烯侧链绕过磷酸化中间体的 ATP 依赖性形成。