2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation.
The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli-based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6β-hydroxylase activity, possesses a 2β-hydroxylase activity towards DOC and androstenedione as well as a 16β-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6β-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties.
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
A
A
Mosa
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
J
J
Neunzig
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
A
A
Gerber
Institute of Pharmaceutical Biology, Saarland University, 66123 Saarbrücken, Germany.
J
J
Zapp
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
F
F
Hannemann
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.
P
P
Pilak
Institute of Biochemistry, Saarland University, 66123 Saarbrücken, Germany. Electronic address: ritabern@mx.uni-saarland.de.
R
R
Bernhardt
D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003687:Dehydroepiandrosterone / Q000378:metabolism; D003900:Desoxycorticosterone / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology*; D004970:Estrone / Q000378:metabolism / Q000494:pharmacology*; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011284:Pregnenolone / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D012996:Solutions; D004970:Estrone; D013739:Testosterone; D000735:Androstenedione; D003900:Desoxycorticosterone; D003687:Dehydroepiandrosterone; D004958:Estradiol; D011284:Pregnenolone; D002786:Cholesterol Side-Chain Cleavage Enzyme
CYP11A1; Cholesterol; Cytochromes P450; Steroid 2β,-6β- and 16β-hydroxylation; Steroids; Whole-cell system