Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.     

Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns

To answer the question whether the most common allelic variants of human CYP1A1, namely CYP1A1.1 (wild type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn), differ in their catalytic activity towards eicosapentaenoic acid (EPA), in vitro enzymatic assays were performed in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed EPA epoxygenation and hydroxylation to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr) and 19-OH-EPA, yet with varying catalytic efficiency and distinct regiospecificity. CYP1A1.1 and CYP1A1.4 formed 17(R),18(S)-EETeTr as main product (K(m)=53 and 50 microM; V(max)=0.60 and 0.50 pmol/min/pmol; V(max)/K(m)=0.11 and 0.10 microM(-1)min(-1), respectively), followed by 19-OH-EPA (K(m)=76 and 93 microM; V(max)=0.37 and 0.37 pmol/min/pmol; V(max)/K(m)=0.005 and 0.004 microM(-1)min(-1), respectively). The variant CYP1A1.2 produced almost equal amounts of both metabolites, but its catalytic efficiency for hydroxylation was five times higher (K(m)=66 microM; V(max)=1.7 pmol/min/pmol; V(max)/K(m)=0.026 microM(-1)min(-1)) and that for epoxygenation was twice higher (K(m)=66 microM; V(max)=1.5 pmol/min/pmol; V(max)/K(m)=0.023 microM(-1)min(-1)) than those of the wild-type enzyme. Thus, the Ile462Val polymorphism in human CYP1A1 affects EPA metabolism and may contribute to interindividual variance in the local production of physiologically active fatty acid metabolites in the cardiovascular system and other extrahepatic tissues, where CYP1A1 is expressed or induced by polycyclic aromatic hydrocarbons and other xenobiotics.     

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.     

Purification and characterization of rat sterol 14-demethylase P450 (CYP51) expressed in Escherichia coli.

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.     

Amino acid 305 determines catalytic center accessibility in CYP3A4

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.     

Expression of cytochrome P450 2A6 in Escherichia coli: purification, spectral and catalytic characterization, and preparation of polyclonal antibodies.

Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines. In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture. Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal amino acid sequence of recombinant CYP2A6 was blocked. The N-terminal formylmethionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K(m) = 1.48 +/- 0.37 microM, V(max) = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the role of CYP2A6 in the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of this enzyme.     

Allelic variation in the Depressaria pastinacella CYP6AB3 protein enhances metabolism of plant allelochemicals by altering a proximal surface residue and potential interactions with cytochrome P450 reductase

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V(max) of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V(max) of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val(92) on the proximal face of CYP6AB3v1 with Ala(92) in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants.     

Purification of recombinant wheat cytochrome P450 monooxygenase expressed in yeast and its properties

To investigate the properties of wheat cytochrome P450 and the characteristics of herbicide metabolism by cytochrome P450 in vitro, deeply understand the mechanisms of herbicide selectivity, recombinant wheat cytochrome P450 monooxygenase (CYP71Cv1) heterologously expressed in yeast was purified by DE-52 cellulose chromatography and fast protein liquid chromatography (FPLC) with Mono-Q column. The degree of purification was 1366-fold. The specific activity of purified cytochrome P450 reached to 512 nmol min-1 mg-1 protein with herbicide chlorsulfuron as substrate. The purified cytochrome P450 exhibited one band in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular mass was 52.5 kDa. Kinetic parameter was determined in vitro. The Km values for chlorsulfuron and triasulfuron were 57 (+/-15) and 38 (+/-16) microM, respectively; and Vmax for chlorsulfuron and triasulfuron were 4.1 (+/-0.7) and 2.7 (+/-0.5) nmol min-1 mg-1protein in vitro, respectively.     

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.     

Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.     

Catalytic turnover of pyrene by CYP3A4: evidence that cytochrome b5 directly induces positive cooperativity

The metabolism of pyrene to hydroxypyrene by CYP3A4 was investigated to determine the effect of cytochrome b5 (b5) on turnover kinetics. In the absence of b5, formation of hydroxypyrene in in vitro incubations showed a biphasic substrate-velocity curve where K(m1) and V(max1) were 1.3 microM and 0.5 pmol/min/pmol P450, respectively. The addition of testosterone to the incubation mixture completely abolished the second phase to yield a typical, hyperbolic curve, presumably through the disruption in the formation of a pi-pi stacked pyrene complex within the CYP3A4 active site. Finally, the addition of b5 yielded an increase hydroxypyrene formation that resulted in a sigmoidal substrate velocity curve. The V(max) was 15.7 pmol/min/pmol P450, the K(m) was 7.5 microM, and the Hill coefficient was greater than two. This demonstrated that b5 could directly induce positive cooperativity on CYP3A4 and that this biological factor needs to be carefully considered when included in in vitro P450 reactions.     

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

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.     

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.     

In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam

Fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile) is a highly active, broad spectrum insecticide from the phenyl pyrazole family, which targets the gamma-amino butyric acid (GABA) receptor. Although fipronil is presently widely used as an insecticide and acaricide, little information is available with respect to its metabolic fate and disposition in mammals. This study was designed to investigate the in vitro human metabolism of fipronil and to examine possible metabolic interactions that fipronil may have with other substrates. Fipronil was incubated with human liver microsomes (HLM) and several recombinant cytochrome P450 (CYP) isoforms obtained from BD Biosciences. HPLC was used for metabolite identification and quantification. Fipronil sulfone was the predominant metabolite via CYP oxidation. The K(m) and V(max) values for human liver microsomes are 27.2 microM and 0.11 nmol/mg proteinmin, respectively; for rat liver microsomes (RLM) the K(m) and V(max) are 19.9 microM and 0.39 nmol/mg proteinmin, respectively. CYP3A4 is the major isoform responsible for fipronil oxidation in humans while CYP2C19 is considerably less active. Other human CYP isoforms have minimal or no activity toward fipronil. Co-expression of cytochrome b(5) (b(5)) is essential for CYP3A4 to manifest high activity toward fipronil. Ketoconazole, a specific inhibitor of CYP3A4, inhibits 78% of the HLM activity toward fipronil at a concentration of 2 microM. Oxidative activity toward fipronil in 19 single-donor HLMs correlated well with their ability to oxidize testosterone. The interactions of fipronil and other CYP3A4 substrates, such as testosterone and diazepam, were also investigated. Fipronil metabolism was activated by testosterone in HLM but not in CYP3A4 Supersomes. Testosterone 6beta-hydroxylation in HLM was inhibited by fipronil. Fipronil inhibited diazepam demethylation but had little effect on diazepam hydroxylation. The results suggest that fipronil has the potential to interact with a wide range of xenobiotics or endogenous chemicals that are CYP3A4 substrates and that fipronil may be a useful substrate for the characterization of CYP3A4 in HLM.     

Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1

Benzene is an occupational and environmental toxicant. The main human health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome p450 enzyme system. The cytochrome p450 enzymes CYP2E1 and CYP2F2 are the major contributors to the bioactivation of benzene in rats and mice. Although benzene metabolism has been shown to occur with mouse and human lung microsomal preparations, little is known about the ability of human CYP2F to metabolize benzene or the lung cell types that might activate this toxicant. Our studies compared bronchiolar derived (BEAS-2B) and alveolar derived (A549) human cell lines for benzene metabolizing ability by evaluating the roles of CYP2E1 and CYP2F1. BEAS-2B cells that overexpressed CYP2F1 and recombinant CYP2F1 were also evaluated. BEAS-2B cells overexpressing the enzyme CYP2F1 produced 47.4 +/- 14.7 pmols hydroxylated metabolite/10(6) cells/45 min. The use of the CYP2E1-selective inhibitor diethyldithiocarbamate and the CYP2F2-selective inhibitor 5-phenyl-1-pentyne demonstrated that both CYP2E1 and CYP2F1 are important in benzene metabolism in the BEAS-2B and A549 human lung cell lines. The recombinant expressed human CYP2F1 enzyme had a K(m) value of 3.83 microM and a V(max) value of 0.01 pmol/pmol p450 enzyme/min demonstrating a reasonably efficient catalysis of benzene metabolism (V(max)/K(m) = 2.6). Thus, these studies have demonstrated in human lung cell lines that benzene is bioactivated by two lung-expressed p450 enzymes.     

Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase

To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information.     

Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.     

CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.