PROGESTERONE 16-ALPHA-HYDROXYLASE ACTIVITY IS CATALYZED BY HUMAN CYTOCHROME-P450 17-ALPHA-HYDROXYLASE

Progesterone and pregnenolone are metabolized to 17alpha-hydroxysteroids by a cytochrome P450-dependent 17alpha-hydroxylase (P450c17). The same enzyme can also catalyze the removal of the side-chain of these 17alpha-hydroxylated steroids to yield androstenedione and dehydro-epiandrosterone, 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 17alpha-hydroxyprogesterone as well as 16alpha-hydroxyprogesterone, but no detectable androstenedione was produced. However, pregnenolone was converted to 17alpha-hydroxypregnenolone and, ultimately, dehydroepiandrosterone. No 16alpha-hydroxypregnenolone was produced. The kinetics of progesterone metabolism by the enzyme expressed in COS 1 cells indicated that both 17alpha- and 16alpha-hydroxylated products were produced from a common active site. Microsomes prepared from fetal adrenal and adult testis converted progesterone to 17alpha-hydroxyprogesterone as well as 16alpha-hydroxyprogesterone. No detectable androstenedione was produced by these preparations. Antibodies raised against porcine cytochrome P450c17 inhibited the 17alpha- and 16alpha-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 16alpha-hydroxylase activity in addition to its 17alpha-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, 17alpha-hydroxyprogesterone, and 16alpha-hyclroxyprogesterone and fails to convert these to corresponding C19 steroids.