EPR spectrometry of cytochrome P450 2B4: effects of mutations and substrate binding.

The EPR spectra of NH(2)-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g approximately 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g approximately 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g approximately 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.

Promoter function and the role of cytokines in the transcriptional regulation of rabbit CYP2E1 and CYP2E2.

Rabbit CYP2E1 and CYP2E2 show considerable similarity in the 5' flanking region, but a 32-base-pair element (32-BPE) that is repeated in 2E1 is present only as a single inexact copy in 2E2. In the present investigation, footprinting disclosed two specific binding sites for liver nuclear proteins, and the DNase I sensitivity profiles of the two genes were found to be different. Several positive and negative regulatory elements were identified by transfection with a series of constructs of upstream CYP2E sequences fused to the luciferase gene. Both genes have an HNF-1 consensus motif with one nucleotide mismatch, which affects binding affinity and promoter activity. Investigation of DNA-protein interactions revealed that Sp1 and NFkappaB bind exclusively to the 32-BPE of 2E1 and 2E2, respectively, suggesting a possible regulatory role for the 32-BPE. Interleukin-1alpha (IL-1alpha) gave rise to a 2.5-fold increase in the promoter activity of 2E1 in HepG2 cells, and the IL-1alpha-mediated induction of reporter gene expression was almost completely prevented when the 32-BPE was deleted. Increased DNA binding and Sp1 protein content as a result of IL-1alpha treatment, as well as cotransfection experiments with pPacSp1, suggest that Sp1 is a transcription activator for the induction of 2E1 by IL-1alpha in HepG2 cells.

Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes.

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytochrome P450BM3-F87G results in the covalent modification of the heme cofactor of this monooxygenase. Analysis of the resulting heme by electronic absorption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiacetal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was observed that replacement of the Phe-87 in the P450BM3 by the smaller glycyl residue was essential for the modification to proceed, as the wild-type enzyme showed no spectral changes under identical conditions. The kinetics of this reaction were examined by stopped-flow spectrophotometry with 3-phenylpropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the process of heme modification was biphasic, with initial bleaching of the Soret absorbance, followed by an increase in absorbance centered at 430 nm, consistent with meso-heme adduct formation. The intermediate formed during phase I also showed an increased absorbance between 700 and 900 nm, relative to the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for phase II. All of these observations are consistent with a mechanism involving radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of which produces the gamma-meso-alkyl heme derivative.

Discrete species of activated oxygen yield different cytochrome P450 heme adducts from aldehydes.

Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.

Multiple activated oxygen species in P450 catalysis: contributions To specificity in drug metabolism.

A hypervalent iron-oxene species has been widely proposed as the "active oxygen" in cytochrome P450 (P450)-catalyzed reactions. We recently examined the effect of mutation of the highly conserved threonine residue in P450s 2B4 and 2E1 to alanine, a change that is believed to interfere with proton delivery to the active site, and have determined the change in rates of deformylation of aldehydes, epoxidation of olefins, and hydroxylation of various substrates. The results support the concept that three distinct oxidants are functional in P450 catalysis: nucleophilic peroxo-iron, nucleophilic or electrophilic hydroperoxo-iron, and electrophilic oxenoid-iron. The occurrence of multiple oxidizing species may contribute to the remarkable versatility of the P450 family of isozymes in the modification of drugs and other substrates. Furthermore, the relative concentrations of these oxidants in a particular P450 isozyme may contribute to substrate specificity and govern the type of reaction catalyzed.

Membrane topology of cytochrome P450 2B4 in Langmuir-Blodgett monolayers.

Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring. The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer. Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics. Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion. To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface. The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices. The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S. J., Larson, J. R., Philpot, R. M., and Coon, M. J. (1993) Proc. Natl. Acad. Sci. USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane.

Cytochrome P450 2E1 mRNA in the rat prostate: detection and quantitation by competitive reverse transcription and polymerase chain reaction.

Cytochrome P450 2E1 plays a pivotal role in the metabolic activation of a wide variety of low molecular weight environmental toxicants and procarcinogens. In the present study, expression of the P450 2E1 gene in the rat prostate gland was quantitated by competitive reverse transcription and the polymerase chain reaction. To assess accurately the induction level of P450 2E1 mRNA in the prostate after pyridine treatment of rats, a recombinant standard RNA was generated that is homologous to the sequence of P450 2E1 mRNA except for an internal deletion of 100 bases. The data indicate that P450 2E1 mRNA is present in the prostate of untreated animals and is induced about four-fold by treatment with pyridine. The results suggest that exposure to certain environmental chemicals and procarcinogens may increase P450 2E1 levels in the prostate gland and thus could enhance formation of reactive, carcinogenic metabolites.

Regulation of rabbit cytochrome P450 2E1 expression in HepG2 cells by insulin and thyroid hormone.

The regulation of cytochrome P450 (CYP) 2E1, the ethanol-inducible isoform, is particularly complex. The level is affected by a variety of other foreign compounds, by insulin (as studied in several laboratories), and by triiodothyronine (T3), which has not been previously examined at the molecular level. In the present investigation, a stably transfected HepG2 cell line harboring a rabbit CYP2E1 minigene containing the coding sequence together with 1.6 kilobases of the 5' flanking region and the untranslated region (UTR), as well as 0.5 kilobases of the 3' UTR, was established. Western blot analysis showed that 1 microM insulin decreased the CYP2E1 protein level in a dose- and time-dependent manner, whereas 1 microM T3 increased the level 2-fold in 1 day and 8-fold in 5 days. Similarly, steady state CYP2E1 mRNA levels were decreased by insulin but were increased by T3. Neither hormone affected the transcription rate of the CYP2E1 5' flanking region with an UTR/luciferase fusion gene, indicating that the regulation is post-transcriptional in this system under our experimental conditions. When the CYP2E1 3' UTR was removed from the minigene, CYP2E1 mRNA and protein were up-regulated by insulin but were not affected by T3. These findings, including mRNA half-life determinations, indicate that the effects of insulin and T3 are a result of altered mRNA stability and that the 3' UTR of CYP2E1 contains regulatory information for these hormone-mediated effects.

Characterization of the cytochrome P450 CYP2J4: expression in rat small intestine and role in retinoic acid biotransformation from retinal.

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.

Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant.

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

Metabolic activation of trans-4-hydroxy-2-nonenal, a toxic product of membrane lipid peroxidation and inhibitor of P450 cytochromes.

Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of which trans-4-hydroxy-2-nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two pathways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanism-based inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanism-based inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme.

Mechanism-based inactivation of cytochrome P450 2B4 by aldehydes: relationship to aldehyde deformylation via a peroxyhemiacetal intermediate.

The inactivation of cytochrome P450 2B4 by aldehydes in a reconstituted enzyme system requires molecular oxygen and NADPH and is not prevented by the addition of catalase, superoxide dismutase, epoxide hydrolase, glutathione, or ascorbic acid. A strong correlation between loss of enzymatic activity and bleaching of the heme chromophore was established, and the inactivation was shown to be irreversible upon dialysis. In general, saturated aldehydes are more inhibitory than those with alpha,beta-unsaturation, as indicated by the k(inact) values, and primary aldehydes are more potent inactivators than the structurally related secondary and tertiary aldehydes. Consistent with recent studies on catalytic specificity of the T302A mutant of this cytochrome [Vaz, A. D. N., Pernecky, S. J., Raner, G. M., & Coon, M. J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4644-4648], the rate of aldehyde deformylation, as determined by formation of the alcohol with one less carbon atom, is greatly stimulated over that of the wild-type enzyme. Of particular interest, the rate of oxidation of aldehydes to carboxylic acids is decreased with the mutant, whereas the rate of inactivation via heme destruction is enhanced. Furthermore, comparative deuterium isotope effects and the relative rates of inactivation and product formation suggest that the mechanism of aldehyde inactivation of P450 2B4 involves the deformylation reaction and is unrelated to carboxylic acid formation. Finally, in the reaction of P450 2B4 with 3-phenylpropionaldehyde, the formation of a heme adduct with a molecular weight corresponding to that of native heme plus 104 mass units confirms the loss of the carbonyl group from the aldehyde prior to reaction with the chromophore. We conclude that inactivation of P450 by aldehydes occurs via homolytic cleavage of a peroxyhemiacetal intermediate to give an alkyl radical that reacts with the heme.

Evidence for a role of a perferryl-oxygen complex, FeO3+, in the N-oxygenation of amines by cytochrome P450 enzymes.

Most cytochrome P450 (P450)-catalyzed reactions are believed to involve an FeO3+ intermediate as the actual oxygenating species. However, studies on the mechanism of steroid aromatization and subsequent model work have provided evidence that a peroxo-iron form (formally FeO2) can be involved directly in some oxidations. The possible involvement of peroxoiron was considered in P450-catalyzed N-oxygenations, because there is precedent for the use of H2O2 and organic peroxides in such reactions in the literature concerning synthetic and flavin reactions. The approach used was to compare P450 reactions involving the normal NADPH/NADPH-P450 reductase/O2 system with those supported by the oxygen surrogates H2O2 (which can directly form FeO2 and subsequently FeO3+) and iodosylbenzene (which can form FeO3 but not FeO2+). Iodosylbenzene was effective in supporting rabbit P450 1A2-catalyzed N,N-dimethyl-2-aminofluorene N-oxygenation, human P450 3A4-catalyzed quinidine N-oxygenation, rat P450 2B1-catalyzed oxidation of N-benzyl-(1-phenyl) cyclobutylamine to the N-hydroxyamine and nitrone, and rat P450 2B1-catalyzed and rabbit P450 2B4-catalyzed N-oxygenation of N,N-dimethylaniline (also N-demethylation). H2O2 also supported most of these reactions. A mutant of P450 2B4 with the substitution of alanine for threonine at position 302 has been shown to have decreased ability to catalyze reactions involving the putative FeO3+ but, presumably because of decreased ability to protonate the FeO2+ complex, to have enhanced activity in oxidative deformylation reactions believed to involve FeO2+. This mutant showed both decreased N,N-dimethylaniline N-demethylation and N-oxygenation activity. Although some contribution of an FeO2+ species to these reactions cannot be ruled out, formation of product in the iodosylbenzene-supported systems cannot be readily explained by an obligatory FeO2 mechanism and the involvement of FeO3+ is concluded to be more likely.

Metabolic activation of 2,6-dichlorobenzonitrile, an olfactory-specific toxicant, by rat, rabbit, and human cytochromes P450.

The herbicide 2,6-dichlorobenzonitrile (DCBN) is known to cause tissue-specific toxicity at very low doses in the olfactory mucosa of rodents. The toxicity of DCBN is reportedly cytochrome P450 (P450) dependent, but the isoforms involved have not been identified, and the effects of this agent on humans are not known. In the present study, DCBN metabolism was examined with microsomes and with purified P450s in a reconstituted system. Rat and rabbit olfactory microsomes act on DCBN to form DCBN-protein adducts as well as two metabolite peaks, designated M1 and M2, identified through high performance liquid chromatography with radiometric detection. The activity of rat olfactory microsomes in DCBN metabolism is much higher than that of liver or lung microsomes. Of seven purified rabbit P450s known to be expressed in the olfactory mucosa, including 1A2, 2A10/11, 2B4, 2E1, 2G1, and 3A6, the 2A10/11 preparation is the most active, producing M2 as well as DCBN-protein adducts; P450 2E1 is the only other active isoform. The addition of purified epoxide hydrolase (EC 4.2.1.63) to the reconstituted enzyme system leads to the formation of M1 and decreased formation of M2. It seems that M1 and M2 are derived from an epoxide intermediate that also forms covalent protein adducts. Gas chromatography- and liquid chromatography-mass spectrometry analyses of nasal microsomal DCBN metabolites and DCBN-glutathione conjugates indicated that the major reactive intermediate may be 2,3-oxo-DCBN and that M1 may be 2,3-dihydroxy-6-chlorobenzonitrile, whereas M2 may correspond to a monohydroxy-DCBN. Interestingly, heterologously expressed human P450s 2A6 and 2E1, but not 1A2, are active in the metabolism of DCBN, forming protein adducts as well as M2. Thus, the preferential expression of P450s of the 2A subfamily in olfactory tissue suggests a molecular basis for the tissue-specific toxicity of the herbicide and may have important implications for risk assessment in humans.

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Cytochrome P450 2: peroxidative reactions of diversozymes.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive beta-cleavage to give hydrocarbons and aldehydic acids. One of these products, trans-4-hydroxynonenal, inactivates P450, particularly alcohol-inducible 2E1, in what may be a negative regulatory process. Although a P450 iron-oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron-peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.

Metabolism of all-trans, 9-cis, and 13-cis isomers of retinal by purified isozymes of microsomal cytochrome P450 and mechanism-based inhibition of retinoid oxidation by citral.

The involvement of a series of microsomal cytochrome P450 (P450) isozymes in all-trans-retinoid metabolism, including the conversion of all-trans-retinal to all-trans-retinoic acid, was previously described. In the current study, we examined the role of seven liver microsomal P450 isozymes in the oxidation of three isomers of retinal. P450 1A1, which was not tested previously, is by far the most active in the conversion of all-trans-, 9-cis-, and 13-cis-retinal to the corresponding acids, as well as in the 4-hydroxylation of all-trans- and 13-cis retinal. In contrast, P450s 2B4 and 2C3 are the most active in the 4-hydroxylation of 9-cis-retinal, with turnover numbers approximately 7 times as great as that of P450 1A1. The inclusion of cytochrome b5 in the reconstituted enzyme system is without effect or inhibitory in most cases but stimulates the 4-hydroxylation of 9-cis-retinal by P450 2B4, giving a turnover of 3.7 nmol of product/min/nmol of this isozyme, the highest for any of the retinoid conversions we have studied. Evidence was obtained for two additional catalytic reactions not previously attributed to P450 oxygenases: the oxidation of all-trans- and 9-cis-retinal to the corresponding 4-oxo derivatives by isoform 1A2, and the oxidative cleavage of the acetyl ester of vitamin A (retinyl acetate) to all-trans-retinal, also by isoform 1A2. The physiological significance of the latter reaction, with a Km for the ester of 32 microM and a Vmax of 18 pmol/min/nmol of P450, remains to be established. We also examined the effect on P450 of citral, a terpenoid alpha, beta-unsaturated aldehyde and a known inhibitor of cytosolic retinoid dehydrogenases. Evidence was obtained that citral is an effective mechanism-based inactivator of isozyme 2B4, with a KI of 44 microM as determined by the oxidation of 1-phenylethanol to acetophenone, and by isozyme 1A2 in the oxidation of all-trans-retinal to the corresponding acid and by isozyme 2B4 in the 4-hydroxylation of all-trans-retinol and retinoic acid. Thus, citral is not suitable for use in attempts to distinguish between retinoid conversions catalyzed by dehydrogenases in the cytoplasm and by P450 cytochromes in the endoplasmic reticulum.

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.