title: The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.
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 Kmand decreased kcatvalue 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.
authors.affiliation: Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Neunzig
authors.affiliation: Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Milhim
authors.affiliation: Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Schiffer
authors.affiliation: Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Khatri
authors.affiliation: Institute of Pharmaceutical Biology, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Zapp
authors.affiliation: Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus Liebig University, 35392 Giessen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Sánchez-Guijo
authors.affiliation: Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus Liebig University, 35392 Giessen, Germany.
authors.forename: M F
authors.initials: MF
authors.lastname: Hartmann
authors.affiliation: Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetology, Center of Child and Adolescent Medicine, Justus Liebig University, 35392 Giessen, Germany.
authors.forename: S A
authors.initials: SA
authors.lastname: Wudy
authors.affiliation: Department of Biochemistry, Faculty of Technical and Natural Sciences III, Saarland University, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.
authors.forename: R
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000728:Androgens / Q000378:metabolism; D000735:Androstenedione / Q000031:analogs & derivatives* / Q000378:metabolism; D001141:Aromatase / Q000378:metabolism*; D047072:Aromatase Inhibitors / Q000737:chemistry; D002384:Catalysis; D002675:Child, Preschool; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004703:Endocrine System; D004700:Endocrine System Diseases / Q000175:diagnosis / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011944:Receptors, Androgen / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism
chemical_list: C410798:AR protein, human; D000728:Androgens; D047072:Aromatase Inhibitors; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D013256:Steroids; D000735:Androstenedione; C018202:16-hydroxyandrost-4-en-3,17-dione; D001141:Aromatase; C561410:CYP19A1 protein, human; C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase
keywords: 16(beta)- OH-androstenedione; CYP19A1; CYP21A2; LC–MS/MS; Steroidogenesis; androstenedione