Mammalian Cytochrome P450 Research Articles

NMR‐based Structural Model of Rabbit Cytochrome P450 2B4 with the Analgesic Amidopyrine, the Intermediate Desmethylamidopyrine, and its Product Aminoantipyrine: Implications for the Structural Basis of Sequential Metabolism

Rabbit cytochrome P450 2B4 (CYP2B4) and the analgesic amidopyrine were used as prototypes of sequential drug metabolism by mammalian cytochromes P450 (CYPs). CYP2B4 metabolizes amidopyrine by two N‐demethylation steps to form the product didesmethylamidopyrine. Despite numerous studies of amidopyrine with this enzyme, little is known about the structural basis of sequential metabolism. In order to narrow our gaps in understanding, NMR studies were undertaken to build a structural model of binding of amidopyrine, the singly demethylated intermediate, and its product to CYP2B4. Heme‐induced relaxation of ligand protons was used to position the ligands in the active site of CYP2B4. Saturation transfer and NOE experiments were used to probe the protein and aqueous environments of the ligands, respectively. This information was combined and then used as constraints for a molecular dynamics simulation of ligands bound to CYP2B4. Implications of the model will be discussed in the context of sequential metabolism by CYPs as a whole and how this technology can be applied to other CYPs. (Supported by NIH grant ES003619).

Mammalian cytochrome CYP2E1 triggered differential gene regulation in response to trichloroethylene (TCE) in a transgenic poplar

Trichloroethylene (TCE) is an important environmental contaminant of soil, groundwater, and air. Studies of the metabolism of TCE by poplar trees suggest that cytochrome P450 enzymes are involved. Using poplar genome microarrays, we report a number of putative genes that are differentially expressed in response to TCE. In a previous study, transgenic hybrid poplar plants expressing mammalian cytochrome P450 2E1 (CYP2E1) had increased metabolism of TCE. In the vector control plants for this construct, 24 h following TCE exposure, 517 genes were upregulated and 650 genes were downregulated over 2-fold when compared with the non-exposed vector control plants. However, in the transgenic CYP2E1 plant, line 78, 1,601 genes were upregulated and 1,705 genes were downregulated over 2-fold when compared with the non-exposed transgenic CYP2E1 plant. It appeared that the CYP2E1 transgenic hybrid poplar plants overexpressing mammalian CYP2E1 showed a larger number of differentially expressed transcripts, suggesting a metabolic pathway for TCE to metabolites had been initiated by activity of CYP2E1 on TCE. These results suggest that either the over-expression of the CYP2E1 gene or the abundance of TCE metabolites from CYP450 2E1 activity triggered a strong genetic response to TCE. Particularly, cytochrome p450s, glutathione S-transferases, glucosyltransferases, and ABC transporters in the CYP2E1 transgenic hybrid poplar plants were highly expressed compared with in vector controls.

Oxidative and Conjugative Xenobiotic Metabolism in Zebrafish Larvae In Vivo

As zebrafish larvae are being increasingly applied to toxicity testing, there is a need to understand the potential for xenobiotic metabolism by these early life-stage organisms. The expression of genes similar to mammalian cytochromes P450 (CYP) 2B6, CYP3A5, and UDP-glucuronosyl transferase (UGT) 1A1, as well as the zebrafish CYP1A was assessed across embryonic development. Activities toward 7-ethoxyresorufin O-deethylase (EROD assay), 7-ethoxycoumarin O-deethylase (ECOD assay), and octyloxymethylresorufin (OOMR assay) were detected at 96 h postfertilization, as was significant phenolic conjugation in the EROD assay (p < 0.001). The induction of CYP1A, the CYP gene zgc:153269, and UGT1A1 after exposure to Aroclor 1254 (100 microg/L, 24 h) was observed, with significant CYP1A induction (p < 0.01). Aroclor exposure also significantly induced EROD activity (p < 0.005), as did coexposure of alpha-naphthoflavone in a dose-dependent manner (p < 0.05; 5 and 10 microM exposures). Inhibition of CYP activity by SKF525A (5 microM) could not be demonstrated because of significant CYP induction as evidenced by OOMR activity (p < 0.05). This study demonstrates that zebrafish larvae express genes similar to mammalian CYP and UGT isoforms throughout early development and have activities toward model CYP substrates. The modulation of these genes and activities by CYP inducers is also reported. The continued use of these model organisms in toxicity testing is supported by this study.

Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0-Å Resolution

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

Exhaustive computational search of ionic-charge clusters that mediate interactions between mammalian cytochrome P450 (CYP) and P450-oxidoreductase (POR) proteins

In this work, a model for the interaction between CYP2B4 and the FMN domain of rat P450-oxidoreductase is built using as template the structure of a bacterial redox complex. Amino acid residues identified in the literature as cytochrome P450 (CYP)–redox partner interfacial residues map to the interface in our model. Our model supports the view that the bacterial template represents a specific electron transfer complex and moreover provides a structural framework for explaining previous experimental data. We have used our model in an exhaustive search for complementary pairs of mammalian CYP and P450-oxidoreductase (POR) charge clusters. We quantitatively show that among the previously defined basic clusters, the 433K–434R cluster is the most dominant (32.3% of interactions) and among the acidic clusters, the 207D–208D–209D cluster is the most dominant (29%). Our analysis also reveals the previously not described basic cluster 343R–345K (16.1% of interactions) and 373K (3.2%) and the acidic clusters 113D–115E–116E (25.8%), 92E–93E (12.9%), 101D (3.2%) and 179E (3.2%). Cluster pairings among the previously defined charge clusters include the pairing of cluster 421K–422R to cluster 207D–208D–209D. Moreover, 433K–434R and 207D–208D–209D, respectively the dominant positively and negatively charged clusters, are uncorrelated. Instead our analysis suggests that the newly identified cluster 113D–115E–116E is the main partner of the 433K–434R cluster while the newly described cluster 343R–345K is correlated to the cluster 207D–208D–209D.

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.

Functional Characterization of the re-Face Loop Spanning Residues 536−541 and Its Interactions with the Cofactor in the Flavin Mononucleotide-Binding Domain of Flavocytochrome P450 from Bacillus megaterium

Flavocytochrome P450BM-3, a bacterial monooxygenase, contains a flavin mononucleotide-binding domain bearing a strong structural homology to the bacterial flavodoxin. The flavin mononucleotide (FMN) serves as the one-electron donor to the heme iron, but in contrast to the electron transfer mechanism of mammalian cytochrome P450 reductase, the FMN semiquinone state is not thermodynamically stable and appears transiently as the anionic rather than the neutral form. A unique loop region comprised of residues (536)Y-N-G-H-P-P(541), which forms a type I' reverse turn and provides several interactions with the FMN isoalloxazine ring, was targeted in this study. Nuclear magnetic resonance studies support the presence of a strong hydrogen bond between the backbone amide of Asn537 and FMN N5, the anionic ionization state of the hydroquinone, and for a change in the hybridization state of the N5 upon reduction. Replacement of Tyr536, which flanks the flavin ring, with a basic residue (histidine or arginine) did not significantly influence the redox properties of the FMN or the accumulation of the anionic semiquinone. The central residues of the type I' turn (Asn-Gly) were replaced with various combinations of glycine and alanine as a means of altering the turn and its interactions. Gly538 was found to be crucial in maintaining the type I' turn conformation of the loop and the strong H-bonding interaction at N5. The functional role of the tandem Pro-Pro sequence which anchors and possible "rigidifies" the loop was investigated through alanine replacements. Despite changes in the stabilities of the oxidized and hydroquinone redox states of the FMN, none of the replacements studied significantly altered the two-electron midpoint potentials. Pro541 does contribute to some degree to the strength of the N5 interaction and the formation of the anionic semiquinone. Unlike that of the flavodoxin, it would appear that the conformation of the FMN rather than the loop changes in response to reduction in this flavoprotein.

13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a (13)CH(3)-reporter attached. This (13)C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site.

Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

The ability of four mammalian cytochromes P450 (CYP) of the CYP1A subfamily, human and mouse CYP1A1s and human and rabbit CYP1A2s, to metabolize a series of steroids and related compounds was investigated us- ing high throughput approaches. Oxidation rates and metabolite patterns for 16 steroid substrates and for 20 polycyclic aromatic hydrocarbon (PAH) substrates were determined in standardized automated conditions. Multivariate statistics of normalized activity data sets was used to sort out significant information and to compare functional signatures of assayed enzymes. Interestingly, for steroid substrates, rabbit CYP1A2 unambiguously aggregates with human and mouse CYP1A1s and appears functionally divergent from human CYP1A2. In contrast, the functional classification was found consistent with the sequence classification when exogenous PAH substrates were tested. The observed features rely on a large set of substrates, all presenting a similar chemical scaffold but decorated with different substituents similar to chemical series used in drug development. Differential functional clusters are thus evidenced for endogenous and exoge- nous substrates with CYP1A enzymes. A few residues on rabbit CYP1A2 that may account for its unusual 1A1-like speci- ficity toward steroids have been identified both within the active site and at the protein surface. These specific residues thus seem to play a controlling role for global substrate class discrimination, potentially by involving substrate bulkiness and shape sensing.

Metabolic Activation of Mifepristone [RU486; 17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)-estra-4,9-dien-3-one] by Mammalian Cytochromes P450 and the Mechanism-Based Inactivation of Human CYP2B6

Mifepristone [RU486; 17beta-hydroxy-11beta-(4-dimethylaminophenyl)-17alpha-(1-propynyl)-estra-4,9-dien-3-one] inactivates CYP2B6 in the reconstituted system in a mechanism-based manner. The loss of 7-ethoxy-4-(trifluoromethyl)-coumarin deethylation activity of CYP2B6 is concentration- and time-dependent. The inactivation requires NADPH and is irreversible. The concentration of inactivator required to give the half-maximal rate of inactivation is 2.8 microM, and the maximal rate constant for inactivation at a saturating concentration of the inactivator is 0.07 min(-1). Incubation of CYP2B6 with 20 microM RU486 for 15 min resulted in 61% loss of catalytic activity, 60% loss of the reduced cytochrome P450 (P450)-CO complex, and a 40% loss of native heme. The partition ratio is approximately 5, and the stoichiometry of binding is approximately 0.6 mol RU486/mol P450 inactivated. SDS-polyacrylamide gel electrophoresis and high-pressure liquid chromatography analysis showed that [(3)H]RU486 was irreversibly bound to CYP2B6 apoprotein. RU486 is metabolized to form three major metabolites and bioactivated to give reactive intermediates by purified P450s in the reconstituted system. After incubation of RU486 with the purified P450s and liver microsomes from rats and humans in the presence of glutathione (GSH) and NADPH, GSH conjugates with MH(+) ions at m/z 769, 753, and 751 were detected by liquid chromatography-tandem mass spectrometry. Two GSH conjugates with MH(+) ions at m/z 753 are formed from the reaction of GSH with RU486. The adducts are formed after addition of an activated oxygen to the carbon-carbon triple bond of the propynyl moiety. This suggests that oxirene intermediates may be involved in the mechanism of inactivation. It seems that the potential for drug-drug interactions of RU486 may not be limited only to CYP3A4 and should also be evaluated for drugs metabolized primarily by CYP2B6, such as bupropion and efavirenz.

Chiral Polychlorinated Biphenyls Are Biotransformed Enantioselectively by Mammalian Cytochrome P-450 Isozymes to Form Hydroxylated Metabolites

Chiral Polychlorinated Biphenyls Are Biotransformed Enantioselectively by Mammalian Cytochrome P-450 Isozymes to Form Hydroxylated Metabolites

Endogenous Functions of the Aryl Hydrocarbon Receptor (AHR): Intersection of Cytochrome P450 1 (CYP1)-metabolized Eicosanoids and AHR Biology

The AHR,2 a ligand-activated transcription factor, was first identified indirectly in the 1970s; the mouse and human genes were cloned in the early 1990s. Molecular mechanisms and biological consequences of AHR-mediated regulation of mammalian cytochrome P450 enzymes by foreign chemicals (e.g. polycyclic aromatic hydrocarbons and dioxins) have been studied extensively. Binding of such ligands to AHR leads to transcriptional activation of CYP1A1, CYP1A2, and CYP1B1; in turn, these enzymes catalyze oxidative detoxication or activation of most ligands. From the start, AHR endogenous ligands and functions were postulated; although this was initially controversial, the robust phenotype of Ahr–/– knock-out mice provided clear evidence of physiological roles (and endogenous ligands) for AHR. AHR has numerous important endogenous functions: during conception and embryonic and fetal development; in the immune, cardiovascular, neural, and reproductive systems; and in hepatocytes, skin cells, and adipocytes. These myriad AHR-mediated processes mirror the vast universe of action of the eicosanoids, lipid mediators known to undergo cytochrome P450-dependent oxidation. We propose that many endogenous and exogenous cellular stimuli lead to (i) AHR-dependent CYP1-dependent eicosanoid synthesis and degradation and (ii) AHR-dependent CYP1-independent (eicosanoid-dependent and -independent) responses. These two pathways can be delineated from one another in genetic models: the former is absent in the recently characterized Cyp1a1/1a2/1b1–/– triple-knock-out mouse, and the latter is absent in the Ahr–/– knock-out mouse. Identification of specific eicosanoids whose synthesis or degradation is carried out by each CYP1 enzyme should allow for identification of physiological endogenous AHR ligands. (For “History and Background,” see supplemental material (Box 1).)

Chiral Polychlorinated Biphenyls Are Biotransformed Enantioselectively by Mammalian Cytochrome P-450 Isozymes to Form Hydroxylated Metabolites

In vitro incubations of purified rat cytochrome P-450 (CYP) 2B1 and human CYP 2B6 were performed to determine if CYP isozymes biotransform polychlorinated biphenyls (PCBs) enantioselectively. Enantioselective metabolism of chiral PCBs 45, 84, 91, 95, 132, and 136 and production of hydroxylated PCB metabolites (OH-PCBs) were observed, while no changes in PCB 183 atropisomer composition were observed for either isozyme. Enantiomer fractions (EFs) of parent PCBs, individually incubated as racemates at 25 ng/mL initial concentration, with rat CYP 2B1 ranged from 0.353 to 0.822. Enantioselectivity was also observed for PCBs 45 (EF = 0.437) and 132 (EF = 0.537) incubated at that concentration with human CYP 2B6. Both atropisomers of chiral PCBs appeared to be biotransformed simultaneously by rat CYP 2B1, except for (+)-PCB 132, but at different rates. Hydroxylated PCBs were identified using gas chromatography-high resolution mass spectrometry for all chiral PCBs enantioselectively transformed by CYPs. These metabolites did not correspond to any commercially available authentic standards, supporting the hypothesis that many unidentified OH-PCBs detected in wildlife may have arisen from in vivo biotransformation of chiral PCBs. A rough estimate suggested that more than half of the total congener metabolized by rat CYP 2B1 was converted to OH-PCBs. Similar concentration decreases were observed for congeners incubated with human CYP 2B6, but less OH-PCBs were formed. Formation of OH-PCBs via an enantioselective OH insertion mechanism was suggested, and may be a source of the unidentified OH-PCBs currently found in the environment.

New findings in studies of cytochromes P450

Cytochromes P450 represent a numerous family of heme-containing enzymes belonging to the group of monooxygenases. In prokaryotes, cytochromes P450 usually perform a plastic function, whereas in eukaryotes their functions are very diverse. Mammalian cytochromes P450 are components of membranes and are involved in biosynthesis and metabolism of many physiologically active substances; moreover, these cytochromes are unique in their ability to catalyze biotransformation of xenobiotics, i.e. metabolize substances of foreign origin (drugs, toxins, environmental pollutants). The latter promotes elimination of xenobiotics, but sometimes intermediates of their metabolism are even more toxic and dangerous than the original xenobiotics per se. Some catalytic features of cytochromes P450 still need unambiguous explanation, i.e. broad substrate specificity, diversity of catalytic reactions, and unusual kinetics. Under some conditions, cytochromes P450 can produce reactive oxygen species, and this is another problem attracting increasing attention. In this respect, a recent finding in mitochondria of analogs of microsomal cytochromes P450 seems especially intriguing; it was postulated that P450 can be responsible for mitochondrial dysfunction, cell apoptosis, and pathogenesis of some diseases. In this paper the present state of the art concerning these problems is considered.

Metabolism of Phenylahistin Enantiomers by Cytochromes P450: A Possible Explanation for Their Different Cytotoxicity

Phenylahistin is a fungal diketopiperazine derived from isoprenylated (Phe-DeltaHis) cyclodipeptide. The (-)-enantiomer is a cell cycle inhibitor, which can be potentially used as an antitumor agent. By contrast, the (+)-enantiomer exhibits no antimicrotubule activity. To better understand the differences that could arise from a difference of bioavailability, we investigated the interaction and metabolism of both enantiomers with mammalian cytochromes P450 (P450s). We found that both enantiomers were metabolized by various isoforms of mammal P450 with a noticeable activity for the (+)-enantiomer. P450 3A isoforms were mainly responsible for this metabolism, the bioactive (-)-enantiomer being 1.5 to 8 times less metabolized than the (+)-enantiomer. Spectral analysis of the interaction with P450s revealed that (-)-phenylahistin led to a hydrophobic type I signature, whereas the (+)-isomer yielded a Fe-N type II one. Structural analysis of metabolites by liquid chromatography-tandem mass spectrometry allowed us to characterize two major metabolites (P1 and P3) for both enantiomers. In human liver microsomal preparations, P1 was predominant in the (-)-phenylahistin metabolic profile. In contrast, (+)-phenylahistin mainly produced P3 in human microsomes and CYP3A human expressed P450s. (-)-Phenylahistin proved to be less toxic on P450-rich hepatocytes than on P450-deprived KB lines. The slower metabolism of this enantiomer could account for its higher toxicity. This is strengthened by the fact that isolated metabolites of (-)-phenylahistin showed no toxic effects toward KB lines. Finally, differences of metabolism and interaction mode between both phenylahistin enantiomers and CYP3A4 were supported by in silico molecular docking calculations.

Biocatalysis and substrate chemodiversity: Adaptation of aerobic living organisms to their chemical environment

A unique family of proteins, the cytochromes P450, catalyze the oxidation of almost all the compounds of our environment, often called xenobiotics. They play a key role in the adaptation of aerobic living organisms to their always changing chemical environment. How a single family of catalysts can operate in a relatively efficient manner on such extremely diverse substrates? Recent X-ray structures of mammalian xenobiotic-metabolizing P450s (mainly from human liver) that have been published during the 2003–2007 period, allow one to begin to answer this question. These data have shown the great diversity of sizes, shapes, and modes of binding of the substrate binding sites of these mammalian cytochromes P450. They have also shown how conformationally flexible are these active sites, that can adapt themselves to the xenobiotic structure for the best possible efficacy of substrate oxidation catalysis.

Transgenic plants for phytoremediation: helping nature to clean up environmental pollution

Phytoremediation is the use of plants to clean up environmental pollution. However, detoxification of organic pollutants by plants is often slow, leading to the accumulation of toxic compounds that could be later released into the environment. A recent publication by Doty and colleagues describes the development of transgenic poplars (Populus) overexpressing a mammalian cytochrome P450, a family of enzymes commonly involved in the metabolism of toxic compounds. The engineered plants showed enhanced performance with regards to the metabolism of trichloroethylene and the removal of a range of other toxic volatile organic pollutants, including vinyl chloride, carbon tetrachloride, chloroform and benzene. This work suggests that transgenic plants might be able to contribute to the wider and safer application of phytoremediation.

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6‐drug interactions

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes.

Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans

The genome of Caenorhabditis elegans contains 75 full length cytochrome P450 (CYP) genes whose individual functions are largely unknown yet. We tested the hypothesis that some of them may be involved in the metabolism of eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of this nematode. Microsomes isolated from adult worms contained spectrally active CYP proteins and showed NADPH-CYP reductase (CPR) activities. They metabolized EPA and with lower activity also arachidonic acid (AA) to specific sets of regioisomeric epoxy- and ω-/(ω-1)-hydroxy-derivatives. 17( R),18( S)-epoxyeicosatetraenoic acid was produced as the main EPA metabolite with an enantiomeric purity of 72%. The epoxygenase and hydroxylase reactions were NADPH-dependent, required the functional expression of the CPR-encoding emb-8 gene, and were inhibited by 17-ODYA and PPOH, two compounds known to inactivate mammalian AA-metabolizing CYP isoforms. Multiple followed by single RNAi gene silencing experiments identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to EPA metabolism in C. elegans. Liquid chromatography/mass spectrometry revealed that regioisomeric epoxy- and hydroxy-derivatives of EPA and AA are endogenous constituents of C. elegans. The endogenous EPA metabolite levels were increased by treating the worms with fenofibrate, which also induced the microsomal epoxygenase and hydroxylase activities. These results demonstrate for the first time that C. elegans shares with mammals the capacity to produce CYP-dependent eicosanoids and may thus facilitate future studies on the mechanisms of action of this important class of signaling molecules.

Thermodynamic Fidelity of the Mammalian Cytochrome P450 2B4 Active Site in Binding Substrates and Inhibitors

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (ΔΔ G) of ∼ 0.5 to 1 kcal/mol compared with the wild type but with a large entropy–enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a ΔΔ G of ∼ 0.5 kcal/mol but with as much as 40 kcal/mol of entropy–enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a ΔΔ G of ∼ 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.