Cholesterol is a key sterol whose homeostasis is primarily maintained through bile acid metabolism. Proper bile acid formation is vital for nutrient and fat-soluble vitamin absorption and emulsification of lipids. Synthesis of bile acids occurs through two main pathways, both of which rely on 3-hydroxy-5-C27 steroid oxidoreductase (HSD3B7) to begin epimerization of the 3β hydroxyl of cholesterol into its active 3α conformation. In this sequence HSD3B7 catalyzes the dehydrogenation of the 3β-hydroxy group followed by isomerization of the Δ5-cholestene-3-one. These reactions are some of the many steps that transform cholesterol for either storage or secretion. HSD3B7 has distinct activity from other 3β-HSD family members leaving significant gaps in our understanding of its mode of catalysis and substrate specificity. Additionally, the role of HSD3B7 in health and disease positions it as a metabolic vulnerability that could be harnessed as a therapeutic target. To this end, we evaluated the mechanism of HSD3B7 catalysis and reveal that HSD3B7 displays activity towards diverse 7α-hydroxylated oxysterols. HSD3B7 retains its catalytic efficiency towards these substrates, suggesting that its substrate binding pocket can withstand changes in polarity upon alterations to this hydrocarbon tail. Experiments aimed at determining substrate order are consistent with HSD3B7 catalyzing a sequential ordered bi bi reaction mechanism with the binding of NAD+ followed by 7α-hydroxycholesterol to form a central complex. HSD3B7 bifunctional activity is dependent on membrane localization through a putative membrane-associated helix giving insight into potential regulation of enzyme activity. We found strong binding of the NADH product thought to activate the isomerization reaction. Homology models of HSD3B7 reveal a potential substrate pocket that allows for oxysterol binding and mutagenesis was utilized to support this model. Together these studies offer an understanding of substrate specificity and kinetic mechanism of HSD3B7 which can be exploited for future drug development.

中文翻译：

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3-羟基-Δ5-C27-类固醇氧化还原酶的底物特异性和动力学机制。


胆固醇是一种关键的甾醇，其稳态主要通过胆汁酸代谢来维持。适当的胆汁酸形成对于营养和脂溶性维生素的吸收和脂质的乳化至关重要。胆汁酸的合成通过两个主要途径进行，这两个途径都依赖于 3-羟基-5-C27 类固醇氧化还原酶 （HSD3B7） 开始胆固醇的 3β 羟基差向异构化为其活性的 3α 构象。在这个序列中，HSD3B7 催化 3β-羟基的脱氢，然后是 Δ5-胆甾烯-3-酮的异构化。这些反应是转化胆固醇以储存或分泌胆固醇的众多步骤中的一部分。HSD3B7 具有与其他 3β-HSD 家族成员不同的活性，这在我们对其催化模式和底物特异性的理解中留下了重大空白。此外，HSD3B7 在健康和疾病中的作用使其成为一种代谢脆弱性，可以用作治疗靶点。为此，我们评估了 HSD3B7 催化的机制，并揭示了 HSD3B7 对多种 7α-羟基化氧甾醇表现出活性。HSD3B7 保持了对这些底物的催化效率，表明其底物结合袋可以承受该碳氢化合物尾部改变时的极性变化。旨在确定底物顺序的实验与 HSD3B7 一致，HSD3B7 催化顺序有序的 bi bi 反应机制，NAD+ 随后与 7α-羟基胆固醇结合形成中央复合物。HSD3B7 双功能活性依赖于通过推定的膜相关螺旋的膜定位，从而深入了解酶活性的潜在调节。我们发现 NADH 产物的强结合被认为会激活异构化反应。 HSD3B7 的同源模型揭示了一个潜在的底物口袋，该口袋允许氧甾醇结合和诱变，用于支持该模型。这些研究共同提供了对 HSD3B7 的底物特异性和动力学机制的理解，可用于未来的药物开发。