Progesterone 16 alpha-hydroxylase activity is catalyzed by human cytochrome P450 17 alpha-hydroxylase.
Progesterone and pregnenolone are metabolized to 17 alpha-hydroxysteroids by a cytochrome P450-dependent 17 alpha-hydroxylase (P450c17). The same enzyme can also catalyze the removal of the side-chain of these 17 alpha-hydroxylated steroids to yield androstenedione and dehydroepiandrosterone, respectively. We investigated the metabolism of progesterone by monkey kidney tumor (COS 1) cells transfected with a plasmid vector containing the cDNA encoding the complete amino acid sequence for human cytochrome P450c17. Transfected COS 1 cells converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone, but no detectable androstenedione was produced. However, pregnenolone was converted to 17 alpha-hydroxypregnenolone and, ultimately, dehydroepiandrosterone. No 16 alpha-hydroxypregnenolone was produced. The kinetics of progesterone metabolism by the enzyme expressed in COS 1 cells indicated that both 17 alpha- and 16 alpha-hydroxylated products were products were produced from a common active site. Microsomes prepared from fetal adrenal and adult testis converted progesterone to 17 alpha-hydroxyprogesterone as well as 16 alpha-hydroxyprogesterone. No detectable androstenedione was produced by these preparations. Antibodies raised against porcine cytochrome P450c17 inhibited the 17 alpha- and 16 alpha-hydroxylation of progesterone to the same extent when using fetal adrenal microsomes, whereas no inhibition of 21-hydroxylation of progesterone was observed. Similar results were obtained with the imidazole antimycotic agent ketoconazole, which is a preferential cytochrome P450c17 inhibitor. From these results we conclude that human cytochrome P450c17 exhibits marked progesterone 16 alpha-hydroxylase activity in addition to its 17 alpha-hydroxylase function when expressed not only in a heterologous cell expression system but also, importantly, in human steroidogenic cells. Furthermore, the human enzyme has extremely low C-17,20-lyase activity toward progesterone, 17 alpha-hydroxyprogesterone, and 16 alpha-hydroxyprogesterone and fails to convert these to corresponding C19 steroids.
Swart
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Cecil H. and Ida Green Center for Reproductive Biology Sciences, Departments of Biochemistry and Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235.
Swart
A C
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Waterman
M R
MR
Estabrook
R W
RW
Mason
J I
JI
D019326:17-alpha-Hydroxyprogesterone; D000311:Adrenal Glands / Q000196:embryology / Q000648:ultrastructure; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D004247:DNA / Q000235:genetics; D006801:Humans; D006908:Hydroxyprogesterones / Q000378:metabolism; D007654:Ketoconazole / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D039181:Steroid 16-alpha-Hydroxylase; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013737:Testis / Q000648:ultrastructure; D014162:Transfection
D006908:Hydroxyprogesterones; C018975:16-hydroxyprogesterone; D011374:Progesterone; D009249:NADP; D019326:17-alpha-Hydroxyprogesterone; D011284:Pregnenolone; D004247:DNA; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8; D039181:Steroid 16-alpha-Hydroxylase; D013254:Steroid 17-alpha-Hydroxylase; D007654:Ketoconazole