Steroidogenic Cytochromes P450

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Most steroidogenic enzymes are members of the cytochrome P450 group of oxidases (1,2). All of these enzymes have molecular weights about 50 kDa and contain a single heme group. They are called "P450" ("pigment" 450) because they all exhibit a characteristic absorbance at 450 nm upon reduction with carbon monoxide (3). The mechanism by which all the steroidogenic P450s function is identical. They all reduce atmospheric oxygen with electrons from nicotinamide-adenine-dinucleotide phosphate (NADPH). These electrons reach the P450 by one or more protein intermediates. For the mitochondrial P450s, two protein intermediates, adrenodoxin reductase and adrenodoxin, are involved. For the microsomal P450s, one protein intermediate, P450 reductase, is involved.

The adrenal, gonadal, and placental synthesis of all steroid hormones from cholesterol involves six distinct cytochromes P450 (Fig. 1), while the synthesis of neurosteroids involves three cytochromes P450 (Fig. 2). P450scc is the cholesterol side chain cleavage enzyme; P450c17 is 17a hydroxylase/17,20 lyase; P450c21 is 21 hydroxylase; P450c1ip is 11 p hydroxylase, P450c11AS is aldosterone synthase and P450aro is aromatase. P450scc, P450c11|3, and P450c11AS are mitochondrial enzymes; P450c21, P450c17, and P450aro are microsomal enzymes. The synthesis of specific steroid hormones in the adrenal, gonads, and placenta is dependent on the tissue-specific expression of these various enzymes. Thus, P450c11 p and P450c11AS are expressed in the adrenal and not the gonads or placenta, resulting in glucocorticoid and mineralocorticoid production, whereas expression of P450c17 in the testes results in androgen production, and P450aro expression in the gonads results in estrogen production.

As is evident from the reactions shown in Fig. 1, many of the steroid hydroxylases have multiple enzymatic activities. Purification of the proteins and cloning of the cDNAs encoding these proteins has rigorously demonstrated that these activities indeed reside within single proteins.

The synthesis of neurosteroids probably proceeds through pathways both similar to and different from those used in the adrenals, gonads, and placenta. The brain contains additional steroid metabolizing enzymes that convert classic steroid hormones to a variety of neuroactive compounds, shown in Fig. 2. Pregnenolone (PREG), dehydroeplandrosterone (DHEA), and metabolites of progesterone (PROG) and 20a hydroxyprogesterone have been identified in the brains of many species. These metabolites include 5a dihydroprogesterone and 3a,5a-tetrahydroprogesterone (allopregnanolone), suggesting that the brain metabolizes PROG in a fashion different from adrenals and gonads. Just as in the adrenals, gonads, and placenta, the expression of the steroidogenic enzymes in the central and peripheral nervous systems is develop-mentally, regionally, and cell-specifically regulated, ensuring the regulated synthesis of specific neurosteroids.

P450scc

The first, rate-limiting, and hormonally regulated step in the synthesis of all steroid hormones is the conversion of cholesterol to PREG. This reaction is catalyzed the mitochondrial enzyme cholesterol side-chain cleavage, P450scc. The conversion of cholesterol to PREG involves three chemical reactions: 20a hydroxylation, 22 hydroxylation, and scission of the C20 C22 carbon bond. The products of this reaction are pregnenolone

Cholesterol Side Chain Cleavage Enzyme

Fig. 1. Steroid hormone synthesis in "classic" steroidogenic tissues. The names of the enzymes are shown for each reaction. P450scc, cholesterol side-chain cleavage; 3pHSD, 3 p hydroxysteroid dehydrogenase/isomerase; P450c17, 17a hydroxylase/c17,20 lyase; P450c21, 21 hydroxylase; P450cttp, lip hydroxylase; P450c11AS, aldosterone synthase; P450c11B3, developmentally-regulated 11 p hydroxylase/18 hydroxylase; 17PHSD, 17p hydroxysteroid dehydrogenase/17 ke-tosteroid reductase; P450aro, aromatase. "ZF/R" refers to the adrenal zona fasciculata/reticularis and "ZG" refers to the adrenal zona glomerulosa. The numbers next to 3pHSD and 17PHSD refer to the enzyme type that mediates the particular conversion in human beings. P450scc, P450c11p and P450c11AS are mitochondrial enzymes; P450c21, P450c17, and P450aro are microsomal enzymes. The synthesis of specific steroid hormones in the adrenal, gonads and placenta is dependent on the tissue-specific expression of these various enzymes. P450c11p and P450c11AS are expressed in the human and rat adrenal and not in the gonads or placenta, resulting in glucocor-ticoid and mineralocorticoid production; P450c11B3 is developmentally expressed only in the rat adrenal. P450c17 is expressed in the human adrenal (which synthesizes abundant quantities of DHEA and the glucocorticoid cortisol) but not in the rodent adrenal (which synthesizes the glucocorticoid corticosterone, and does not synthesize DHEA). P450c17 is expressed in the rat placenta, but not in the human placenta, and hence the human adrenal provides androgen substrates for estrogen synthesis. P450c17 expression in the testes and ovarian theca cells results in androgen production, and P450aro expression in the ovaries converts androgens into estrogens.

and isocaproic acid. Protein purification and cDNA cloning have demonstrated that a single protein is responsible for these three reactions, and that the same protein is found in all steroidogenic tissue, including the brain (4,5).

P450scc is the rate-limiting step in steroidogenesis, and is one of the slowest enzymes known, with a Vmax of ~1 mol cholesterol/mol enzyme/s. The slowest part of this reaction may be the entry of cholesterol into the mitochondria and binding to the active site of P450scc.

The human and rat genomes contain a single gene encoding P450scc (9) which is about 20 kb long, contains nine exons, and in humans is located on chromosome 15. This gene encodes an mRNA about 2.0 kb, which encodes a 521-amino-acid protein. This protein is proteolytically cleaved, removing a 39-amino-acid leader peptide that directs the protein to the mitochondria.

Sulfotransferase PREGNENOLONE SULFATE ^ ^

Sulfatase

CHOLESTEROL ^P450scc

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