Heterologous expression, purification, and properties of human cytochrome P450 27C1

Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.     

Metabolism of cholesterol, vitamin D3 and 20-hydroxyvitamin D3 incorporated into phospholipid vesicles by human CYP27A1

CYP27A1 is a mitochondrial cytochrome P450 which can hydroxylate vitamin D3 and cholesterol at carbons 25 and 26, respectively. The product of vitamin D3 metabolism, 25-hydroxyvitamin D3, is the precursor to the biologically active hormone, 1α,25-dihydroxyvitamin D3. CYP27A1 is attached to the inner mitochondrial membrane and substrates appear to reach the active site through the membrane phase. We have therefore examined the ability of bacterially expressed and purified CYP27A1 to metabolize substrates incorporated into phospholipid vesicles which resemble the inner mitochondrial membrane. We also examined the ability of CYP27A1 to metabolize 20-hydroxyvitamin D3 (20(OH)D3), a novel non-calcemic form of vitamin D derived from CYP11A1 action on vitamin D3 which has anti-proliferative activity on keratinocytes, leukemic and myeloid cells. CYP27A1 displayed high catalytic activity towards cholesterol with a turnover number (k(cat)) of 9.8 min(-1) and K(m) of 0.49 mol/mol phospholipid (510 μM phospholipid). The K(m) value of vitamin D3 was similar for that of cholesterol, but the k(cat) was 4.5-fold lower. 20(OH)D3 was metabolized by CYP27A1 to two major products with a k(cat)/K(m) that was 2.5-fold higher than that for vitamin D3, suggesting that 20(OH)D3 could effectively compete with vitamin D3 for catalysis. NMR and mass spectrometric analyses revealed that the two major products were 20,25-dihydroxyvitamin D3 and 20,26-dihydroxyvitamin D3, in almost equal proportions. Thus, the presence of the 20-hydroxyl group on the vitamin D3 side chain enables it to be metabolized more efficiently than vitamin D3, with carbon 26 in addition to carbon 25 becoming a major site of hydroxylation. Our study reports the highest k(cat) for the 25-hydroxylation of vitamin D3 by any human cytochrome P450 suggesting that CYP27A1 might be an important contributor to the synthesis of 25-hydroxyvitamin D3, particularly in tissues where it is highly expressed.     

Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli

Bovine adrenodoxin (Adx) was expressed on the surface of Escherichia coli as a monomeric fusion protein with the translocation unit of the AIDA-I autotransporter. The fusion protein remained anchored in the outer membrane by the beta-barrel of the autotransporter. Dimeric Adx molecules were formed spontaneously on the bacterial surface with high efficiencies. Adx dimers could be activated to biological function by chemical incorporation of the [2Fe-2S] cluster. By adding purified adrenodoxin reductase and P450 CYP11A1, a whole cell biocatalyst system was obtained, which effectively synthesized pregnenolone from cholesterol. Addition of artificial membrane constituents or detergents, which was indispensable before to get functional steroidal P450 enzymes, was not necessary. The whole cell activity (0.21 nmol x h(-1) x nmol(-1) CYP11A1) was in the same range as obtained earlier for reconstitution assays. The whole cell system developed here is an easy to handle, stable tool for the expression of membrane-associated P450 enzymes without the need of microsome preparation or reconstitution of artificial membrane vesicles. Moreover, it is the first report on functional dimer formation of a protein anchored on the surface of E. coli after being transported as a monomer. This seems to be a special feature of the autotransporter translocation unit, containing a beta-barrel, motile in the outer membrane and opens a new dimension for the surface display of multimeric proteins.     

25-Hydroxylation of vitamin D3 in primary cultures of pig hepatocytes: evidence for a role of both CYP2D25 and CYP27A1

There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).     

Metabolism of vitamin D(3) by human CYP27A1

Human vitamin D(3) 25-hydroxylase (CYP27A1) cDNA was expressed in Escherichia coli, and its enzymatic properties were revealed. The reconstituted system containing the membrane fraction prepared from the recombinant E. coli cells was examined for the metabolism of vitamin D(3). Surprisingly, at least eight forms of metabolites including the major product 25(OH)D(3) were observed. HPLC analysis and mass spectrometric analysis suggested that those metabolites were 25(OH)D(3), 26(OH)D(3), 27(OH)D(3), 24R,25(OH)(2)D(3), 1alpha, 25(OH)(2)D(3, )25,26(OH)(2)D(3) (25,27(OH)(2)D(3)), 27-oxo-D(3) and a dehydrogenated form of vitamin D(3). These results suggest that human CYP27A1 catalyzes multiple reactions and multiple-step metabolism toward vitamin D(3). The K(m) and V(max) values for vitamin D(3) 25-hydroxylation and 25(OH)D(3) 1alpha-hydroxylation were estimated to be 3.2 microM and 0.27 (mol/min/mol P450), and 3.5 microM and 0.021 (mol/min/mol P450), respectively. These kinetic studies have made it possible to evaluate a physiological meaning of each reaction catalyzed by CYP27A1.     

Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES

The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Identification of a novel rat microsomal vitamin D3 25-hydroxylase

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.     

Physiological role of the N-terminal processed P4501A1 targeted to mitochondria in erythromycin metabolism and reversal of erythromycin-mediated inhibition of mitochondrial protein synthesis

Recently, we showed that the major species of beta-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5-7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8-12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5-6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.     

Adrenodoxin--a versatile ferredoxin

Mammalian adrenodoxin (Adx) has been known for many years as an essential electron mediator in mitochondrial cytochrome P450 systems. Because of its ability to support several cytochrome P450 enzymes, it is involved not only in adrenal steroid hormone biosynthesis but also in vitamin D and bile acid metabolism. Recently, Adx is increasingly gaining attention because of its potential for pharmaceutical industry and biotechnology. With human cytochromes P450 becoming important drug targets, suitable Adx-based screening systems have to be developed to test putative new drugs. Moreover, in artificial systems, Adx has been shown to functionally interact with diverse bacterial cytochromes P450 catalyzing a variety of chemically interesting reactions. Putative biotechnological applications of such Adx-containing reconstituted systems are discussed.     

High-level expression of functional human cytochrome P450 1A2 in Escherichia coli.

Enzymatically active human cytochrome P450 1A2 was expressed in Escherichia coli utilizing the pCWori+ vector containing a modified cDNA. The coding sequence for the NH2-terminal region of the protein was modified by the alignment and substitution of a 27 bp segment from a modified bovine P450 17A1 cDNA onto the 5' end of the open reading frame of P450 1A2 at amino acid 21. The expressed chimeric P450 was produced at a high level in a functionally intact form, as assayed by the formation in vivo of the 449 nm absorbance band of the CO complex of the reduced hemoprotein. E. coli membrane preparations were shown to contain P450 1A2, which was active in the 2-hydroxylation of estradiol, and the O-deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin, when reconstituted with recombinant rat liver NADPH-cytochrome P450 reductase.     

Metabolism of vitamin D by human microsomal CYP2R1

The activation of vitamin D requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney. However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation. Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase. Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D(3) 25-hydroxylase. A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D(2). CYP2R1 hydroxylated vitamin D(2) at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27. The K(m) and k(cat) values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D(3) were 0.45microM and 0.97min(-1), respectively. The k(cat)/K(m) value of CYP2R1 was 26-fold higher than that of CYP27A1. These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans.     

Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin: evaluating site-directed mutations by molecular modeling

The present study was undertaken to evaluate the role of positively charged amino acid residues proposed to reside on the proximal surface of bovine cytochrome P450 cholesterol side chain cleavage (P450scc, CYP11A1) and to determine which residues may be involved in protein-protein interactions with the electron carrier adrenodoxin (Adx). In previous studies, nine different lysine residues were identified by chemical and immunological cross-linking experiments as potentially interacting with Adx, while in the present study, two arginine residues have been identified from sequence alignments. From these 11 residues, 13 different P450scc mutants were made of which only seven were able to be expressed and characterized. Each of the seven mutants were evaluated for their ability to bind Adx, to be reduced, and for their enzymatic activity. Among these, K403Q and K405Q showed a consistent decrease in Adx binding, the ability to be reduced by Adx, and enzymatic activity, with K405Q being affected to a much greater extent. More dramatic was the complete loss of Adx binding by R426Q, while still retaining its ability to be chemically reduced and bind carbon monoxide. Independently, a homology model of P450scc was constructed and docked with the structure of Adx. Four potential sites of interaction were identified: P450scc:K403 with Adx:D76, P450scc:K405 with Adx:D72; P450scc:R426 with Adx:E73, and P450scc:K267 with Adx:E47. Thus, the biochemical and molecular modeling studies together support the hypothesis that K267, K403, K405, and R426 participate in the electrostatic interaction of P450scc with Adx.     

Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1

Cytochrome P450 27A1 (P450 27A1) is an important metabolic enzyme involved in bile acid biosynthesis and the activation of vitamin D3 in mammals. Recombinant P450 27A1 heterologously expressed in Escherichia coli was found to be copurified with phospholipids (PLs). The PL content varied in different preparations and was dependent on the purification protocol. A link between the increased amounts of PLs and deterioration of the enzyme substrate binding properties was also observed. Tandem negative ionization mass spectrometry identified phosphatidylglycerol (PG) as the major PL copurified with P450 27A1. Subsequent reconstitution of P450 into exogenous PG vesicles assessed the effect of this contamination on substrate binding and enzyme activity. Two other PLs, phosphatidylethanolamine (PE) and phosphatidylserine (PS), were also tested. PG and PE increased the Kd for 5beta-cholestane-3alpha,7alpha,12alpha-triol and cholesterol binding, whereas PS had no effect on either substrate binding. PG and PE did not significantly alter 5beta-cholestane-3alpha,7alpha,12alpha-triol hydroxylase activity and even stimulated cholesterol hydroxylase activity. PS inhibited 5beta-cholestane-3alpha,7alpha,12alpha-triol hydrolyase activity and had no effect on cholesterol hydroxylase activity. Our study shows the potential for PLs to regulate the activity of P450 27A1 in vivo and alter the amount of cholesterol degraded through the "classical" and "alternative" bile acid biosynthetic pathways.     

25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria

Mitochondrial cytochrome P450 systems are an indispensable component of mammalian steroid biosynthesis; they catalyze regio- and stereo-specific steroid hydroxylations and consist of three protein entities: adrenodoxin reductase (AdR), adrenodoxin (Adx), and a mitochondrial cytochrome P450 enzyme, e.g., CYP11A1 (P450 side chain cleavage, P450scc). It is known that the latter two are able to generate reactive oxygen species (ROS) in vitro . In this study, we investigated whether this ROS generation also occurs in vivo and, if so, whether it leads to the induction of apoptosis. We found that overexpression of either human or bovine Adx causes a significant loss of viability in 11 different cell lines. This loss of viability does not depend on the presence of the tumor suppressor protein p53. Transient overexpression of human Adx in HCT116 cells leads to ROS production, to a disruption of the mitochondrial transmembrane potential (DeltaPsi), to cytochrome c release from the mitochondria, and to caspase activation. In contrast, the effect of transient overexpression of human CYP11A1 on cell viability varies in different cell lines, with some being sensitive and others not. We conclude that mitochondrial cytochrome P450 systems are a source of mitochondrial ROS production and can play a role in the induction of mitochondrial apoptosis.