Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30-40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17ß-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

Inhibition of human CYP1 enzymes by a classical inhibitor α-naphthoflavone and a novel inhibitor N-(3, 5-dichlorophenyl)cyclopropanecarboxamide: An in vitro and in silico study.

Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1-mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N-(3,5-dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α-naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10-95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100-fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10-680 times higher than the ones of ANF. DCPCC was a mixed-type inhibitor of CYP1A2. ANF was a competitive tight-binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2-mediated metabolism of procarcinogens and anticancer drugs.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes.

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Alpha-naphthoflavone induces apoptosis through endoplasmic reticulum stress via c-Src-, ROS-, MAPKs-, and arylhydrocarbon receptor-dependent pathways in HT22 hippocampal neuronal cells.

α-Naphthoflavone (αNF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of αNF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. αNF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced αNF-induced cell death. αNF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced αNF-induced CHOP expression and cell death. αNF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced αNF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, αNF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced αNF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced αNF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced αNF-induced transcriptional activity of AhR. Taken together, these findings suggest that αNF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells.

Benzoflavones as cholesterol esterase inhibitors: Synthesis, biological evaluation and docking studies.

A library of forty 7,8-benzoflavone derivatives was synthesized and evaluated for their inhibitory potential against cholesterol esterase (CEase). Among all the synthesized compounds seven benzoflavone derivatives (A-7, A-8, A-10, A-11, A-12, A-13, A-15) exhibited significant inhibition against CEase in in vitro enzymatic assay. Compound A-12 showed the most promising activity with IC50 value of 0.78nM against cholesterol esterase. Enzyme kinetic studies carried out for A-12, revealed its mixed-type inhibition approach. Molecular protein-ligand docking studies were also performed to figure out the key binding interactions of A-12 with the amino acid residues of the enzyme's active site. The A-12 fits well at the catalytic site and is stabilized by hydrophobic interactions. It completely blocks the catalytic assembly of CEase and prevents it to participate in ester hydrolysis mechanism. The favorable binding conformation of A-12 suggests its prevailing role as CEase inhibitor.

Identification of CYP1B1-specific candidate inhibitors using combination of in silico screening, integrated knowledge-based filtering, and molecular dynamics simulations.

CYP1B1 is a promising drug target for developing novel drugs against hormonal cancers and hypertension. The development of CYP1B1-specific inhibitors is hindered mainly due to non-specific action of known CYP inhibitors. The active site of CYP1B1 is similar to other cytochromes with different substrate preferences rendering a scope to develop specific inhibitors. We have developed a novel in silico approach for design of selective CYP1B1 inhibitors. The approach consists of deriving details of CYP1B1-specific molecular interactions from prior studies, which is used to perform screening of CYP1B1 with NCI compounds. The conventional compound screening is also complemented with the concept of cutoff distance between heme (Fe) and compounds. The binding free energies and HB percentage occupancy calculations of 94 compounds of cluster 1 have verified the docking results using MD. The docking interactions in the active-site cavity of 7 clusters are also taken into account for optimal binding. Hence, we used knowledgebase filtering and MD simulations to enable discovery of selective CYP1B1 inhibitors. The final filtered lead candidates consist of compounds sandwiched between phenylalanine π-π stacking and less than 6 Å from heme (Fe) for enzymatic action. The findings in the study can help development of novel CYP1B1 selective inhibitors.

Characterization of arachidonic acid metabolism by rat cytochrome P450 enzymes: the involvement of CYP1As.

Cytochrome P450 (P450) enzymes mediate arachidonic acid (AA) oxidation to several biologically active metabolites. Our aims in this study were to characterize AA metabolism by different recombinant rat P450 enzymes and to identify new targets for modulating P450-AA metabolism in vivo. A liquid chromatography-mass spectrometry method was developed and validated for the simultaneous measurements of AA and 15 of its P450 metabolites. CYP1A1, CYP1A2, CYP2B1, CYP2C6, and CYP2C11 were found to metabolize AA with high catalytic activity, and CYP2A1, CYP2C13, CYP2D1, CYP2E1, and CYP3A1 had lower activity. CYP1A1 and CYP1A2 produced ω-1→4 hydroxyeicosatetraenoic acids (HETEs) as 88.7 and 62.7%, respectively, of the total metabolites formed. CYP2C11 produced epoxyeicosatrienoic acids (EETs) as 61.3%, and CYP2C6 produced midchain HETEs and EETs as 48.3 and 29.4%, respectively, of the total metabolites formed. The formation of CYP1A1, CYP1A2, CYP2C6, and CYP2C11 major metabolites followed an atypical kinetic profile of substrate inhibition. CYP1As inhibition by α-naphthoflavone or anti-CYP1As antibodies significantly reduced ω-1→4 HETE formation in the lungs and liver, whereas CYP1As induction by 3-methylcholanthrene resulted in a significant increase in ω-1→4 HETEs formation in the heart, lungs, kidney, and livers by 370, 646, 532, and 848%, respectively. In conclusion, our results suggest that CYP1As and CYP2Cs are major players in the metabolism of AA. The significant contribution of CYP1As to AA metabolism and their strong inducibility suggest their possible use as targets for the prevention and treatment of several diseases.

Identifying cytochrome p450 functional networks and their allosteric regulatory elements.

Cytochrome P450 (CYP) enzymes play key roles in drug metabolism and adverse drug-drug interactions. Despite tremendous efforts in the past decades, essential questions regarding the function and activity of CYPs remain unanswered. Here, we used a combination of sequence-based co-evolutionary analysis and structure-based anisotropic thermal diffusion (ATD) molecular dynamics simulations to detect allosteric networks of amino acid residues and characterize their biological and molecular functions. We investigated four CYP subfamilies (CYP1A, CYP2D, CYP2C, and CYP3A) that are involved in 90% of all metabolic drug transformations and identified four amino acid interaction networks associated with specific CYP functionalities, i.e., membrane binding, heme binding, catalytic activity, and dimerization. Interestingly, we did not detect any co-evolved substrate-binding network, suggesting that substrate recognition is specific for each subfamily. Analysis of the membrane binding networks revealed that different CYP proteins adopt different membrane-bound orientations, consistent with the differing substrate preference for each isoform. The catalytic networks were associated with conservation of catalytic function among CYP isoforms, whereas the dimerization network was specific to different CYP isoforms. We further applied low-temperature ATD simulations to verify proposed allosteric sites associated with the heme-binding network and their role in regulating metabolic fate. Our approach allowed for a broad characterization of CYP properties, such as membrane interactions, catalytic mechanisms, dimerization, and linking these to groups of residues that can serve as allosteric regulators. The presented combined co-evolutionary analysis and ATD simulation approach is also generally applicable to other biological systems where allostery plays a role.

Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone.

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes or Baculosomes containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this system was dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450-P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes™. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450-P450 interactions.

The molecular basis for the inhibition of human cytochrome P450 1A2 by oroxylin and wogonin.

In our previous kinetics studies the natural products oroxylin and wogonin were shown to have strong biological affinity for, and inhibitory effects against, human cytochrome P450 1A2, with IC(50) values of 579 and 248 nM, respectively; this might lead to the occurrence of drug-drug interactions when co-administered clinically. However, their inhibitory mechanisms against 1A2 remain elusive. In this study, molecular docking and molecular dynamics simulations were performed to better understand the molecular basis of their inhibitory mechanisms towards 1A2. Structural analysis revealed that oroxylin has a different binding pattern from wogonin and another very strongly binding inhibitor α-naphthoflavone (ANF, IC(50) = 49 nM). The O(7) atom of oroxylin forms hydrogen bonds with the OD1/OD2 atoms of Asp313, which is not observed in the 1A2-wogonin complex. Because of energetically unfavorable repulsions with the methoxy group at the 6 position of the oroxylin ring, significant conformational changes were observed for the sidechain of Thr118 in the MD simulated model. As a result, the larger and much more open binding-site architecture of the 1A2-oroxylin complex may account for its weaker inhibitory effect relative to the 1A2-ANF complex. Energy analysis indicated that oroxylin has a less negative predicted binding free energy of -19.8 kcal/mol than wogonin (-21.1 kcal/mol), which is consistent with our experimental assays. Additionally, our energy results suggest that van der Waals/hydrophobic and hydrogen-bonding interactions are important in the inhibitory mechanisms of oroxylin whereas the former is the underlying force responsible for strong inhibition by ANF and wogonin.

Hepatic cytochrome P450s attenuate the cytotoxicity induced by leflunomide and its active metabolite A77 1726 in primary cultured rat hepatocytes.

The Black Box Warning section of the U.S. drug label for leflunomide was recently updated to include stronger warnings about potential hepatotoxicity from this novel anti-arthritis drug. Because metabolic activation is a key mechanism for drug-induced hepatotoxicity, we examined whether leflunomide and its major metabolite, A77 1726, are cytotoxic to primary rat hepatocytes and whether their toxicity is modulated by hepatic cytochrome P450s (CYPs). As measured by lactate dehydrogenase leakage, time-dependent cytotoxicity was observed at 250-500 µM for leflunomide and 330-500 µM for A77 1726 within 20 h. Unexpectedly, three nonisoenzyme-specific CYP inhibitors, including SKF-525A, metyrapone, and 1-aminobenzotriazole, did not reduce but remarkably enhanced the cytotoxicity of leflunomide or A77 1726. SKF-525A pretreatment notably rendered hepatocytes susceptible to as low as 15 µM leflunomide or A77 1726. Three isoenzyme-specific CYP inhibitors including alpha-naphthoflavone, ticlopidine, and ketoconazole that mainly target CYP1A, CYP2B/2C, and CYP3A, respectively, also enhanced the cytotoxicity. A strong synergistic effect, similar to SKF-525A alone, was noted using a combination of all three of the isoenzyme-specific inhibitors. Hepatocytes pretreated with the CYP inducer dexamethasone for 24 h exhibited decreased cytotoxicity to leflunomide and A77 1726. At the concentrations tested, the CYP inhibitors and inducer showed no cytotoxicity. These data demonstrate that the parent forms of leflunomide and A77 1726 are more toxic to hepatocytes than their poorly characterized metabolites, indicating that the metabolic process of leflunomide is a detoxification step rather than an initiating event leading to toxicity.

Daily treatment with α-naphthoflavone enhances follicular growth and ovulation rate in the rat.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and the first protein involved in a variety of physiological and toxicological processes, including those of xenobiotic metabolizing enzymes. AhR has been found in the ovary of many species and seems to mediate the ovarian toxicity of many environmental contaminants, which are AhR ligands. However, the role of AhR in the ovarian function is unknown. Therefore, the aim of this work was to study the action of α-naphthoflavone (αNF), known to be an AhR antagonist, on both follicular growth and ovulation. Immature Sprague-Dawley rats were daily injected intraperitoneally with αNF (0.1-80 mg/kg) or vehicle for 12 days, and primed with gonadotrophins (eCG/hCG) to induce follicular growth and ovulation. Ovaries were obtained 20 h after hCG administration. By means of immunohistochemistry, we found that the numbers of primordial, primary and antral follicles were increased in rats treated with 80 mg/kg αNF and that there were no differences with other doses. Likewise, the ovarian weight and the ovulation rate, measured by both number of oocytes within oviducts and corpora lutea in ovarian sections, were increased when the rats received either 1 or 10mg/kg daily. Although further studies are necessary to know the mechanism of action of αNF, it is possible that the different ovarian processes can be differentially responsive to the presence of different levels of αNF, and that the same or different endogenous AhR ligands can be involved in these ovarian processes in a cell type-dependent manner.

Structural characterization of the complex between alpha-naphthoflavone and human cytochrome P450 1B1.

The atomic structure of human P450 1B1 was determined by x-ray crystallography to 2.7 Å resolution with α-naphthoflavone (ANF) bound in the active site cavity. Although the amino acid sequences of human P450s 1B1 and 1A2 have diverged significantly, both enzymes exhibit narrow active site cavities, which underlie similarities in their substrate profiles. Helix I residues adopt a relatively flat conformation in both enzymes, and a characteristic distortion of helix F places Phe(231) in 1B1 and Phe(226) in 1A2 in similar positions for π-π stacking with ANF. ANF binds in a distinctly different orientation in P450 1B1 from that observed for 1A2. This reflects, in part, divergent conformations of the helix B'-C loop that are stabilized by different hydrogen-bonding interactions in the two enzymes. Additionally, differences between the two enzymes for other amino acids that line the edges of the cavity contribute to distinct orientations of ANF in the two active sites. Thus, the narrow cavity is conserved in both P450 subfamily 1A and P450 subfamily 1B with sequence divergence around the edges of the cavity that modify substrate and inhibitor binding. The conservation of these P450 1B1 active site amino acid residues across vertebrate species suggests that these structural features are conserved.

Modulation of cytochrome P450 enzyme system by selected flavonoids.

OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.

Mixing apples and oranges: Analysis of heterotropic cooperativity in cytochrome P450 3A4.

Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme's catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, alpha-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38 microM, respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates.

The binding of lignans, flavonoids and coumestrol to CYP450 aromatase: a molecular modelling study.

Androgens are transformed into aromatic estrogens by CYP450 aromatase in a three-step reaction consuming three equivalents of oxygen and three equivalents of NADPH. Estrogens are substrates for nuclear estrogen receptors (ERs) and play a key role in estrogen-dependent tumour cell formation and proliferation. Natural phytoestrogens are proved to be competitive inhibitors of aromatase enzyme at IC(50) values in micromolar levels. In order to understand the mechanisms involved in the binding of various phytoestrogens, we used our model of CYP450 aromatase to study the binding of phytoestrogens using molecular dynamics simulations with a bound phytoestrogen. The simulation trajectory was analysed to find the essential interactions which take place upon binding and a representative structure of the trajectory was minimized for docking studies. Sets of phytoestrogens, such as lignans, flavonoids/isoflavonoids and coumestrol, were docked into the aromatase active site and the binding modes were studied.

Characterization of a 7,8-benzoflavone double effect on CFTR Cl(-) channel activity.

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of adenosine triphosphate (ATP)-binding cassette (ABC) transporter ATPases. This protein forms a Cl(-) channel with a complex regulation; gene mutations cause cystic fibrosis disease. We investigated the interaction between the protein and the flavone UCCF-029 using the patch-clamp technique in the excised inside-out configuration in order to study the molecular mechanism of action for this potentiator on completely phosphorylated channel (25 U/ml protein kinase A) and a relatively low level of ATP (0.3 mM: ). Low concentrations of UCCF-029 (<50 nM: ) increase the open probability (p (o)), favoring the channel transition to an activated state, while high UCCF-029 (>50 nM: ) levels determine inhibition of the CFTR by a reduction of the total open time. Our data suggest that this drug can potentiate CFTR by binding to a specific site on the nucleotide binding domain, promoting dimer formation. The response of CFTR to variable concentrations of ATP is not modified by application of the potentiator UCCF-029 at either low, activatory, concentration or high, inhibitory, levels. Hence, we conclude that the potentiator may not interfere with binding of ATP but probably acts at an independent site in the protein, interacting directly with CFTR to modulate channel activity.

Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2.

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.

Mechanism of interactions of alpha-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe.

Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into alpha-helix A. The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-(bromoacetyl)-2-(dimethylamino)naphthalene (BADAN), 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and alpha-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of the BADAN-modified enzyme is characterized by an S50 of 18.2 +/- 0.7, compared with the value of 2.2 +/- 0.3 for the ANF-induced spin transition, thus revealing an additional low-affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer.

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.