Progesterone 16 alpha-hydroxylase activity is catalyzed by human cytochrome P450 17 alpha-hydroxylase.

Abstract

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.

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

“…Molecular stimulation studies of CYP17 show that substrate binding occurs with the steroid molecule positioned in a proximity parallel to the haem ring . In the CYP17-CPR complex, closer proximity of the C 17 atom to the attacking iron-oxygen intermediate would favour attack on this carbon atom, although hydroxylation at the C 16 position, which has been reported in some cases, is also possible (Swart et al 1993). An atom positioned too distant for oxygenation attack, as in the case of entiomeric forms of progesterone, regardless of a high-spin state, would serve to inhibit CYP17 activity (Auchus et al 2003).…”

Structures of Human Steroidogenic Cytochrome P450 17A1 with Substrates

“…Although the wild type active site cavity topography suggests that pregnenolone could adopt a similar minor orientation resulting in the generation of 16␣-hydroxypregnenolone, no such product is observed experimentally (16). This could be due to a stronger hydrogen bonding interaction with Asn 202 for pregnenolone compared with progesterone.…”

“…Human CYP17A1 performs the 17,20-lyase reaction much more efficiently on 17␣-hydroxypregnenolone to form the physiologically relevant androgen dehydroepiandrosterone, with metabolism of 17␣-hydroxyprogesterone to the corresponding 17,20-lyase product androstenedione 50-fold lower (16,25), despite similar K d values (Table 2). However, the current structural results suggest a basis for this selectivity.…”

“…1) (14,16). It is known that in other species CYP17A1 with Leu at position 105 generates much less 16␣-hydroxyprogesterone (14) and that an A105L mutation in the human CYP17A1 enzyme decreases progesterone 16␣-hydroxylation and increases its 17␣-hydroxylation (17).…”

Bases Moleculares da Hiperplasia Adrenal Congênita

“…Entretanto, a quebra do carbono na posição 17,20 é 50 vezes mais eficiente para a formação de DHEA do que androstenediona, e depende diretamente da ação do citocromo b5, como foi demonstrado em estudos realizados em diferentes tipos de células e organismos (91,92). O citocromo b5 atua como um cofator alostérico (91), ao contrário do citocromo adrenodoxina redutase que atua por transferência de elétrons.…”