Metabolites of vitamin D3 (D3) (cholecalciferol) are recognized as enzymatically formed chemicals in humans that can influence a wide variety of reactions that regulate a large number of cellular functions. The metabolism of D3 has been extensively studied, and a role for three different mitochondrial cytochrome P450s (CYP24A, CYP27A, and CYP27B1) has been described that catalyze the formation of the 24(OH), 25(OH), and 1(OH) metabolites of D3, respectively. The hormone 1,25-dihydroxyvitamin D3 has been most extensively studied and is widely recognized as a regulator of calcium and phosphorous metabolism. Hydroxylated metabolites of D3 interact with the nuclear receptor and thereby influence growth, cellular differentiation, and proliferation. In this article, we describe in vitro experiments using purified mitochondrial cytochrome P450scc (CYP11A1) reconstituted with the iron-sulfer protein, adrenodoxin, and the flavoprotein, adrenodoxin reductase, and show the NADPH and time-dependent formation of two major metabolites of D3 (i.e., 20-hydroxyvitamin D3 and 20,22-dihydroxyvitamin D3) plus two unknown minor metabolites. In addition, we describe the metabolism of 7-dehydrocholesterol by CYP11A1 to a single product identified as 7-dehydropregnenolone. Although the physiological importance of these hydroxylated metabolites of D3 and their in vivo formation and mode of action remain to be determined, the rate with which they are formed by CYP11A1 in vitro suggests an important role.C ytochrome P450scc (CYP11A1) is a mitochondrial hemeprotein oxygenase of great interest because of its identification as a key enzyme in the metabolism of cholesterol to pregnenolone (1). This reaction is the initial step in the pathway leading to the synthesis of a number of metabolically important steroid hormones. Like other mitochondrial P450s this enzyme requires a minielectron transport chain consisting of the iron-sulfur protein, adrenodoxin (Adr), and the FADcontaining NADPH-Adr reductase (AdrR) for the transfer of reducing equivalents from NADPH to the P450. To date, CYP11A1 has no known natural substrates other then cholesterol. The conversion of cholesterol to pregnenolone (P5) is reported to occur by forming in sequence the monohydroxylated product [22R-(OH)cholesterol] followed by the formation of the dihydroxylated intermediate [20␣, 2 cholesterol] with subsequent carbon-carbon bond cleavage at the C20-C22 position to form the C19 steroid pregnenolone (1-5). It has been difficult to identify these hydroxylated intermediates of cholesterol metabolism during catalysis by CYP11A1 because they are not released from the active site of CYP11A1 during metabolism (1-5). Of interest is the recent report that the oxysterol 22R-(OH)cholesterol plays an important role as a ligand for the liver X receptor, a transcription factor (6).In mammalian tissues 7-dehydrocholesterol (7-DHC) is the direct precursor of cholesterol in the Kandutsch-Russell cholesterol biosynthetic pathway. A deficiency of ⌬7-DHC reductase, the enzyme responsible fo...

The adrenal inner zone antigen (IZA), which reacts specifically with a monoclonal antibody raised against the fasciculata and reticularis zones of the rat adrenal, was previously found to be identical with a protein variously named 25‐Dx and membrane‐associated progesterone receptor. IZA was purified as a glutathione S‐transferase‐fused or His6‐fused protein, and its molecular properties were studied. The UV‐visible absorption and EPR spectra of the purified protein showed that IZA bound a heme chromophore in high‐spin type. Analysis of the heme indicated that it is of the b type. Site‐directed mutagenesis studies were performed to identify the amino‐acid residues that bind the heme to the protein. The results suggest that two Tyr residues, Tyr107 and Tyr113, and a peptide stretch, D99–K102, were important for anchoring the heme into a hydrophobic pocket. The effect of IZA on the steroid 21‐hydroxylation reaction was investigated in COS‐7 cell expression systems. The results suggest that the coexistence of IZA with CYP21 enhances 21‐hydroxylase activity.