Neunzig J, Milhim M, Schiffer L, Khatri Y, Zapp J, Sánchez-Guijo A, Hartmann M F, Wudy S A, Bernhardt R
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
Institute of Pharmaceutical Biology, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
J Steroid Biochem Mol Biol. 2017 Mar;167:182-191. doi: 10.1016/j.jsbmb.2017.01.002. Epub 2017 Jan 5.
The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased K and decreased k value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.
21-羟化酶（CYP21A2）是一种对盐皮质激素和糖皮质激素合成至关重要的类固醇生成酶。据描述，它可通过在C21位进行羟基化反应，将孕酮以及17-羟孕酮分别转化为11-脱氧皮质酮（DOC）和11-脱氧皮质醇（RSS）。在本研究中，我们发现CYP21A2的类固醇底物谱比预期的更广泛。利用由纯化的人CYP21A2和人细胞色素P450还原酶（CPR）组成的体外重组系统，我们证明CYP21A2能够代谢DOC、RSS、雄烯二酮（A4）和睾酮（T）。此外，A4的转化产物通过核磁共振光谱确定了其结构，显示在C16-β位有羟基化。对这种类固醇代谢物16(β)-羟基雄烯二酮（16bOHA4）的雄激素特性进行了研究，并与A4进行了比较。两种类固醇代谢物均显示为人类雄激素受体的弱激动剂。此外，还比较了16bOHA4与芳香化酶（CYP19A1）的相互作用与A4的相互作用，表明C16羟基不影响与CYP19A1的结合。相比之下，动力学参数的测定显示，与A4相比，K值增加而k值降低，导致催化效率降低了2倍。这些发现与我们的对接研究一致，揭示了两种类固醇与血红素铁的结合构象和距离相似。此外，还研究了16bOHA4的产物，推测为16-羟基雌酮（16bOHE1）的雌激素活性，与雌二醇和雌酮相比可忽略不计。最后，在一名11-羟化酶缺乏患者和一名内分泌肿瘤患者中发现了16bOHA4。综上所述，本研究提供了关于类固醇激素生物合成的新信息，并提出了一种检测其他潜在相关新型类固醇代谢物的新方法。
