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J Steroid Biochem Mol Biol. 2017 Mar;167:182-191. doi: 10.1016/j.jsbmb.2017.01.002. Epub 2017 Jan 5.

The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

Author information

1
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
2
Institute of Pharmaceutical Biology, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
3
Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus Liebig University, 35392 Giessen, Germany.
4
Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.

Abstract

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 Km and decreased kcat 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.

KEYWORDS:

16(beta)- OH-androstenedione; CYP19A1; CYP21A2; LC–MS/MS; Steroidogenesis; androstenedione

PMID:
28065637
DOI:
10.1016/j.jsbmb.2017.01.002
[Indexed for MEDLINE]

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