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首页> 外文期刊>The Journal of Steroid Biochemistry and Molecular Biology >Human aldosterone synthase: Recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level
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Human aldosterone synthase: Recombinant expression in E. coli and purification enables a detailed biochemical analysis of the protein on the molecular level

机译：人醛固酮合酶：在大肠杆菌中重组表达和纯化可在分子水平上对蛋白质进行详细的生化分析

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Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the blood pressure and has been reported to play a key role in the formation of arterial hypertension, heart failure and myocardial fibrosis. Aldosterone synthase (CYP11B2) catalyzes the biosynthesis of aldosterone by successive 11β- and 18-hydroxylation followed by an 18-oxidation of 11-deoxycorticosterone and thus comprises an important drug target. For more than 20 years, all attempts to purify recombinant human CYP11B2 in significant amounts for detailed analysis failed due to its hydrophobic nature as a membrane protein. Here, we present the successful expression of the protein in E. coli yielding approx. 90 nmol/l culture, its purification and detailed enzymatic characterization. Biochemical analyses have been performed using in vitro conversion assays which revelead a V max of 238 ± 8 nmol productsmol hCYP11B2/min and a K m of 103 ± 8 μM 11-deoxycorticosterone. Furthermore, binding analyses indicated a very loose binding of the first intermediate of the reaction, corticosterone with a K d value of 115 ± 6 μM whereas for 11-deoxycorticosterone a K d of 1.34 ± 0.13 μM was estimated. Upon substrate conversion of 11-deoxycorticosterone, new intermediates have been identified as 19- and 18-hydroxylated products not described before for the human enzyme. To understand the differences in substrate conversion, we constructed a new homology model based on the 3D structure of CYP11A1, performed docking studies and calculated the activation energy for hydrogen abstraction of the different ligands. The data demonstrated that the 11β-hydroxylation requires much less abstraction energy than hydroxylation at C18 and C19. However, the C18 and C19 hydroxylated products might be of clinical importance. Finally, purified CYP11B2 represents a suitable tool for the investigation of potential inhibitors of this protein for the development of novel drugs against hypertension and heart failure as was shown using ketoconazole.
机译：醛固酮是人类最重要的盐皮质激素，参与血压的调节，据报道在形成动脉高血压，心力衰竭和心肌纤维化中起关键作用。醛固酮合酶（CYP11B2）通过连续的11β-和18-羟基化反应，然后11-脱氧皮质酮的18-氧化作用，催化醛固酮的生物合成，因此是重要的药物靶标。 20多年来，由于其作为膜蛋白的疏水性，所有为纯化重组人CYP11B2进行大量纯化以进行详细分析的尝试均告失败。在这里，我们介绍了该蛋白质在大肠杆菌中的成功表达，产生了约90 nmol / l培养物，其纯化和详细的酶促表征。已使用体外转化试验进行了生化分析，该试验的V max为238±8 nmol产物/ nmol hCYP11B2 / min，K m为103±8μM11-脱氧皮质酮。此外，结合分析表明反应的第一中间体皮质酮的结合非常松散，K d值为115±6μM，而对于11-脱氧皮质酮，估计的K d为1.34±0.13μM。在11-脱氧皮质酮的底物转化后，新的中间体已被鉴定为19-和18-羟基化产物，此前未对人类酶进行过描述。为了了解底物转化的差异，我们基于CYP11A1的3D结构构建了一个新的同源性模型，进行了对接研究，并计算了不同配体的夺氢活化能。数据表明11β-羟基化所需的提取能量比在C18和C19处的羟基化要少得多。但是，C18和C19羟基化产物可能具有临床重要性。最后，纯化的CYP11B2代表一种合适的工具，用于研究该蛋白的潜在抑制剂，以开发抗高血压和心力衰竭的新药，如酮康唑所示。

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