The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo.

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546-9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.

Rational engineering of cytochromes P450 2B6 and 2B11 for enhanced stability: Insights into structural importance of residue 334.

Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques.

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis.

Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon-carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 A resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors' knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors.

The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung.

Biotransformation of Cholesterol and 16α,17α-Epoxypregnenolone and Isolation of Hydroxylase in Burkholderia cepacia SE-1.

The metabolism of cholesterol is critical in eukaryotes as a precursor for vitamins, steroid hormones, and bile acids. Some steroid compounds can be transformed into precursors of steroid medicine by some microorganisms. In this study, the biotransformation products of cholesterol and 16α,17α-epoxypregnenolone produced by Burkholderia cepacia SE-1 were investigated, and a correlative enzyme, hydroxylase, was also studied. The biotransformation products, 7ß-hydroxycholesterol, 7-oxocholesterol, and 20-droxyl-16α,17α-epoxypregn-1,4-dien-3-one, were purified by silica gel and Sephadex LH-20 column chromatography and identified by nuclear magnetic resonance and mass spectroscopy. The hydroxylase was isolated from the bacterium and the partial sequences of the hydroxylase, which belong to the catalases/peroxidase family, were analyzed using MS/MS analyses. The enzyme showed activity toward cholesterol and had a specific activity of 37.2 U/mg of protein at 30°C and pH 7.0.

24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver.

Pig liver mitochondria were found to catalyze 27-, 25- and 24-hydroxylation of cholesterol at relative rates of about 1:0.2:0.04. An apparently homogeneous preparation of pig liver mitochondrial cytochrome P-450-27 was found to catalyze the same three hydroxylations at about the same relative rates when reconstituted with adrenodoxin and adrenodoxin reductase. The 24-hydroxycholesterol formed was shown to consist of one of the two possible stereoisomers. When using specifically deuterium-labeled substrates a significant isotope effect was observed in the case of 24-hydroxylation (KH/KD > 10), but not 25-hydroxylation (KH/KD = 1.1), or 27-hydroxylation (KH/KD = 1.1). The difference between the 24-hydroxylation and the other two hydroxylations may be due to different interactions between cholesterol and the same enzyme, with a resulting difference with respect to the rate-limiting step in the reaction. The physiological significance of the mitochondrial 24-hydroxylation is discussed.

Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes.

The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of NADPH-cytochrome P-450s21 were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes have been reported previously (Hiwatashi, A. and Ichikawa, Y. (1979) Biochim. Biophys. Acta 580, 44-63). The steroid 21-hydroxylase system was reconstituted by cytochrome P-450s21 and NADPH-cytochrome P-450 reductase in the presence of detergent. The substrate specificity of the cytochrome P-450s21 was examined with the reconstituted system. The enzyme system was active in the 21-hydroxylations of 17 alpha-hydroxyprogesterone and progesterone, and the N-demethylation of (+)-benzphetamine. The cytochrome P-450s21 purified to as high as 16-17 nmol per mg protein was an electrophoretically pure glycoprotein. The molecular weight of the cytochrome P-450s21 was estimated to be 47 500 by SDS-polyacrylamide gel electrophoresis. Although cytochrome P-450scc or cytochrome P-45011 beta of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450BPA of bovine liver microsomes, the cytochrome P-450s21 formed an immunoprecipitin line against the antibody to cytochrome P-450BPA in the Ouchterlony double diffusion test.

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors.

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.

25-Hydroxylation of 1 alpha-hydroxyvitamin D-3 in rat and human liver.

1 alpha-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1 alpha-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent Km value of 2 X 10(-5) M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100 degrees C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1 alpha-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1 alpha-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.

Hepatic microsomal activities of cholesterol 7 alpha-hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase in the prairie dog. An animal model for cholesterol gallstone disease.

The prairie dog is a useful experimental animal model for studies of cholesterol gallstone pathogenesis. The unique susceptibility to rapid induction of gallstones solely by feeding of a 1.2% cholesterol diet in this species could result from low levels of hepatic cholesterol 7 alpha-hydroxylase. With optimal assay conditions in hepatic microsomes, a basal specific activity of about 25 pmol/min per g protein was found. Administration of diets containing 1.2% cholesterol or 5% cholestyramine caused hydroxylase levels to increase 60 and 250%, respectively. This response pattern is similar to that observed in other species under the same conditions, indicating that abnormally low basal or inappropriately unresponsive hydroxylase levels are not susceptibility factors unique to this model. With optimal assay conditions for hydroxymethylglutaryl-CoA reductase, a Km of 32.5 microM (S-HMG-CoA) and basal specific activities of between 60 and 175 pmol/min per mg protein were found. Following feeding of either sodium chenodeoxycholate or sodium cholate, in reasonable pharmacologic doses, no suppression of hydroxylase and reductase levels was found. These findings undermine the widely held view that the therapeutic effect of oral chenodeoxycholate in man for cholesterol gallstone dissolution is directly mediated by suppression of the activities of these enzymes.

Purification and properties of cytochrome p-450 (11beta- and 18-hydroxylase) from bovine adrenocortical mitochondria.

We describe an improved procedure for the preparation of a cytochrome P-450 from bovine adrenocortical mitochondria which catalyzes 11beta- and 18-hydroxylation of steroids. The preparation is based upon chromatography on DEAE cellulose which separates the enzyme from the side-chain cleavage P-450, which can also be prepared in highly purified form from the same tissue extracts. The enzyme behaves as a single compound in glycerol density gradients. The enzyme aggregates at protein concentrations greater than 1 mg/ml to a series of forms of various molecular weights. On Sepharose 4B the enzyme shows a molecular weight of 185 000, while on glycerol density gradients a molecular weight of 1 - 10(6) is observed. The subunit molecular weight determined by electrophoresis on polyacrylamide gels with sodium dodecyl sulfate is 47 500 and the protein appears as a single band. The ratio of 11beta-/18-hydroxylase activities does not change significantly during purification and is constant through the protein peak on glycerol density gradients. Since there appears to be only one subunit species, it seems likely that the two hydroxylase activities are catalyzed by one protein.

Studies on cytochrome P-450 (P-450 17 alpha,lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5.

We have reported (Kominami, S., Shinzawa K. and Takemori, S. (1982) Biochem. Biophys. Res. Commun. 109, 916-921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17 alpha-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17 alpha-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450 17 alpha,lyase). P-450 17 alpha,lyase had the maximum activity at pH 6.1 both for 17 alpha-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b5 to the reconstituted system, the optimal pH for 17 alpha-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b5. With the addition of cytochrome b5, both the activities were stimulated above pH 6.3-6.5 and were suppressed below pH 6.3-6.5. These results indicate that cytochrome b5 plays some important role in controlling the dual activities of P-450 17alpha,lyase.

Rabbit liver cytochrome P-450 2B5: high-level expression of the full-length protein in Escherichia coli, purification, and catalytic activity.

Rabbit liver cytochrome P-450 2B5 (P-450 2B5) was expressed in Escherichia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by galactose of protein synthesis in the presence of the heme precursor 5-aminolevulinic acid peaked 72 h after addition to the inducer to yield 108 nmol membrane-bound P-450 2B5 per liter of culture medium. The recombinant enzyme was purified to near homogeneity by a two-column procedure involving chromatography on DE-52 cellulose and hydroxylapatite. The hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced CO-difference spectrum with a Soret band at 451 nm. Second-derivative spectral analysis in the middle-UV region revealed that type I binding of 4-nitroanisole to ferric P-450 2B5 abolished absorption bands ascribable to tyrosine residues within the polypeptide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 reductase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reductase redox couple. The enzyme was catalytically active toward 4-nitroanisole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b5. The recombinant hemoprotein did not catalyze bioactivation of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen. The methods described here should facilitate further studies on the biophysical basis of the complex interactions of P-450 2B5 with its redox partners.

Purification and characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: peroxidase-mediated catechol estrogen formation.

Estrogen-2/4-hydroxylase (E-2/4-H) activity of rabbit hypothalamic tissue was previously found to be localized in the soluble subcellular fraction. In the present study, the enzymatic activity responsible for catechol estrogen formation in this subcellular fraction of the rabbit hypothalamus was purified by ammonium sulfate fractionation, ion exchange chromatography, and chromatofocusing. E-2/4-H activity was found to be associated with a group of hemoproteins with peroxidase activity. The characteristics of this hypothalamic E-2/4-H activity were reestablished in light of a peroxidatic mechanism for catechol estrogen formation. Organic hydroperoxides stimulated E-2/4-H activity, presumably by serving as oxidizing cosubstrate required for peroxidase-mediated reactions. E-2/4-H activity in a 17,500 X g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 micrograms/150 microliter. It displayed a pH optimum of 6 and an apparent Michaelis-Menten constant (Km) of 32 microM with respect to estradiol. The amounts of 4-hydroxyestradiol formed were comparable to those of 2-hydroxyestradiol. The characteristics established in this study for the peroxidase-type E-2/4-H were distinct from those of the particulate, NADPH-dependent 2-hydroxylases found in rat liver and in porcine blastocyst and ovary. These differences provide a basis for differentiating between the two types of enzymatic activity that can lead to catechol estrogen formation in vitro.

Purification of 25-hydroxyvitamin D3 24-hydroxylase from rat kidney mitochondria.

25-Hydroxyvitamin D3 24-hydroxylase was purified to an electrophoretically homogeneous state (Mr = 53,000) from kidney mitochondria of female rats treated with vitamin D3.

Purification from pig kidney of a microsomal cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3.

A cytochrome P450 catalyzing 1 alpha-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney microsomes. The enzyme preparation showed one protein band on gel electrophoresis with apparent M(r) of 52,500 and a specific cytochrome P450 content of 10.7 nmol/mg of protein. The 25-hydroxyvitamin D3 1 alpha-hydroxylase copurified with the vitamin D3 25-hydroxylase during purification. A cytochrome P450 catalyzing 1 alpha-hydroxylation was purified also from liver microsomes. The apparently homogeneous enzyme showed the same catalytic properties and apparent M(r) as the kidney enzyme. The results of the present communication demonstrate the presence in kidney of a previously unknown microsomal 1 alpha-hydroxylase in addition to the assumed specific mitochondrial 1 alpha-hydroxylase. The possibility that microsomal 1 alpha-hydroxylation in pig kidney and liver is catalyzed by the previously described porcine microsomal vitamin D 25-hydroxylase(s) is discussed.

1H NMR T1 relaxation rate study on substrate orientation of fluoromethylanilines in the active sites of microsomal and purified cytochromes P450 1A1 and 2B1.

The present study describes 1H NMR T1 relaxation rate studies on fluoromethylanilines bound to the active sites of microsomal and purified cytochromes P450 1A1 and 2B1. From the data obtained, insights into the average orientation of the substrates with respect to the paramagnetic Fe3+ centre in the cytochromes P450 could be derived. Particular attention was paid to a possible extra relaxation pathway for methyl protons compared to the aromatic protons, due to the rotational motion of the CH3 around the sigma-C-CH3 bond. However, this effect appeared to be minimal and to result in at most a few percent underestimation of the actual distance of the methyl protons to the Fe3+ centre. Furthermore, the data obtained demonstrate that all aromatic protons are at about the same average distance from the paramagnetic centre. The results also demonstrate that the fluromethylanilines are bound to the active sites of cytochromes P450 1A1 and 2B1 in a similar way. A time-averaged orientation of the substrate with the Fe3+ above the aromatic ring, with the pi-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable pi-pi interaction defines the orientation which best fits the results of the present study. Possibilities for a flip-flop rotation around an axis in the plane of the aromatic ring can be included in this picture, as such rotations would still result in a similar average distance of all aromatic protons to the Fe3+ paramagnetic centre. The results obtained also indicate that possible differences in metabolite patterns resulting from conversion of the fluoromethylanilines by different cytochromes P450, especially P450 1A1 and 2B1, are unlikely to be caused by a specific orientation of the substrate imposed by the substrate binding site of the enzyme.

Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system.

The mechanism of the cytochrome P450 2B4 modification by hydrogen peroxide (H2O2) formed as a result of partial coupling of NADPH-dependent monooxygenase reactions has been studied in the monooxygenase system reconstituted from the highly purified microsomal proteins: cytochrome P450 2B4 (P450) and NADPH-cytochrome P450 reductase in the presence of detergent Emulgen 913. It was found, that H2O2-mediated P450 self-inactivation during benzphetamine oxidation is accompanied by heme degradation and apoenzyme modification. The P450 heme modification involves the heme release from the enzyme under the action of H2O2 formed within P450s active center via the peroxycomplex decay. Additionally, the heme lost is destroyed by H2O2 localized outside of enzyme's active center. The modification of P450 apoenzyme includes protein aggregation that may be due to the change in the physico-chemical properties of the inactivated enzyme. The modified P450 changes the surface charge that is confirmed by the increasing retention time on the DEAE column. Oxidation of amino acid residues (at least cysteine) may lead to the alteration into the protein hydrophobicity. The appearance of the additional ionic and hydrophobic attractions may lead to the increase of the protein aggregation. Hydrogen peroxide can initiate formation of crosslinked P450 dimers, trimers, and even polymers, but the main role in this process plays nonspecific radical reactions. Evidence for the involvement of hydroxyl radical into the P450 crosslinking is carbonyl groups formation.

A possible role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.

A mitochondrial cytochrome P450 fraction catalyzing 1 alpha- and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney. The ratio between the 1 alpha- and 27-hydroxylase activities was the same in all purification steps including a side fraction. Attempts to separate the 1 alpha- and 27-hydroxylase activities were unsuccessful. A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M(r) and immunoprecipitated both the 1 alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25-hydroxyvitamin D3 hydroxylases in kidney mitochondria. Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1 alpha-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 in kidney. In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.

Cytochrome P450 2C9 is responsible for hydroxylation of the naphthoquinone antimalarial drug 58C80 in human liver.

2-(4-t-Butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone (58C80) is an experimental naphthoquinone antimalarial drug which undergoes extensive alky hydroxylation in man. By means of purification, N-terminal amino acid sequencing and inhibition by antibodies and sulfaphenazole, we have identified the form of cytochrome P450 primarily responsible for 58C80 hydroxylation in human liver, P450hB20-27, to be a member of the P450 2C9 subfamily. P450hB20-27 is a low-spin haemoprotein with molecular mass 54 kDa. 58C80 hydroxylation in human liver microsomes was dependent on either NADPH or NADH, with the activity supported by NADH being 35% of that supported by NADPH. With purified P450hB20-27 cytochrome b5 stimulated the NADH-dependent activity 8-fold but inhibited the NADPH-dependent activity by 30%. 58C80 is a novel substrate structure for human P450 2C and these results significantly broaden the range of drugs which have been directly shown (i.e. using a purified enzyme as opposed to expressed cDNA) to be metabolized by human P450 2C forms that are incontrovertibly expressed in human liver in vivo.