Substrate specificity and kinetic mechanism of 3-hydroxy-Δ5-C27-steroid oxidoreductase.

IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Sarah M Gardner, Austin Vogt, Trevor M Penning, Ronen Marmorstein
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引用次数: 0

Abstract

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.

3-hydroxy-Δ5-C27-steroid oxidoreductase 的底物特异性和动力学机制。
胆固醇是一种重要的固醇,其平衡主要通过胆汁酸代谢来维持。胆汁酸的正常形成对营养物质和脂溶性维生素的吸收以及脂质的乳化至关重要。胆汁酸的合成主要通过两种途径,这两种途径都依赖于 3-hydroxy-5-C27 类固醇氧化还原酶(HSD3B7)开始将胆固醇的 3β 羟基二聚化为其活性的 3α 构象。在这一过程中,HSD3B7 催化 3β- 羟基脱氢,然后将 Δ5- 胆甾烯-3-酮异构化。这些反应是将胆固醇转化为储存或分泌物质的众多步骤中的一部分。HSD3B7 的活性与其他 3β-HSD 家族成员截然不同,因此我们对其催化方式和底物特异性的了解还存在很大差距。此外,HSD3B7 在健康和疾病中的作用将其定位为可用作治疗靶点的代谢弱点。为此,我们评估了 HSD3B7 的催化机制,发现 HSD3B7 对多种 7α- 羟基氧基甾醇具有活性。HSD3B7 对这些底物保持了催化效率,这表明其底物结合袋可以承受碳氢化合物尾部改变后极性的变化。旨在确定底物顺序的实验表明,HSD3B7 催化的是一种顺序有序的双生物反应机制,先是与 NAD+ 结合,然后与 7α- 羟基胆固醇结合,形成一个中心复合物。HSD3B7 的双功能活性依赖于通过一个假定的膜相关螺旋进行的膜定位,这给酶活性的潜在调控带来了启示。我们发现,被认为能激活异构化反应的 NADH 产物有很强的结合力。HSD3B7 的同源模型揭示了一个潜在的底物口袋,它允许氧甾醇结合,我们利用诱变来支持这一模型。这些研究共同提供了对 HSD3B7 底物特异性和动力学机制的理解,可用于未来的药物开发。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Biological Chemistry
Journal of Biological Chemistry Biochemistry, Genetics and Molecular Biology-Biochemistry
自引率
4.20%
发文量
1233
期刊介绍: The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.
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