RESUMEN
CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Polimorfismo de Nucleótido Simple , Empalme Alternativo , Western Blotting , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Mutación del Sistema de Lectura , Expresión Génica , Frecuencia de los Genes , Marcadores Genéticos , Alemania , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Microsomas Hepáticos/enzimología , Fenotipo , Alineación de Secuencia , Análisis de Secuencia de ADN , Suiza , Transcripción Genética , Población Blanca/genéticaRESUMEN
The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released from the membrane using high salt conditions, and were purified from this fraction to specific contents up to 19 nmol P450/mg protein using a single Ni(2+)-agarose column. Gel filtration revealed that truncated P450 2B1 forms a monodisperse solution of hexamers in the absence of detergent and >95% monomers in 0.25% sodium cholate. All truncated proteins, including human 2B6, were active with 7-ethoxy-4-trifluoromethylcoumarin, and truncated 2B1 was shown to retain the native regio- and stereospecificity of testosterone hydroxylation. These data demonstrate that modification of the N-terminus yields high levels of properly folded P450s 2B with increased solubility, which are suitable for functional and structural analysis.
Asunto(s)
Secuencia de Aminoácidos/fisiología , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Expresión Génica , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Eliminación de Secuencia , Animales , Catálisis , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/genética , Perros , Activación Enzimática/fisiología , Humanos , Oxidorreductasas N-Desmetilantes/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Conejos , Ratas , Solubilidad , Relación Estructura-ActividadRESUMEN
At least two substitutions were made at each of five amino acid residues in rat cytochrome P450 2B1 that align to residues of known importance in other P450s. The mutants were histidine tagged for purification from Escherichia coli, and the proteins were assessed for testosterone and 7-alkoxycoumarin oxidation. Alteration of each of the sites studied, Phe-115, Ser-294, Phe-297, Ala-298, and Leu-362, was found to affect overall enzyme activity or the metabolite profile. In particular, most of the mutants, excluding F297A, A298G, and L362F, exhibited significantly altered ratios of 16alpha-hydroxytestosterone:16beta-hydroxytestosterone, with the most dramatic alteration being displayed by A298V. Four 7-butoxycoumarin metabolites were produced by CYP2B1, of which two, 7-hydroxycoumarin and 7-(3-hydroxybutoxy)coumarin, were formed at nearly equal rates. Several mutants, F115A, F297A, F297I, and A298V, exhibited an increased predominance of one of the metabolites. The results from this study illustrate the conservation of functionally important residues across P450 subfamilies and families.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Cumarinas/química , Cumarinas/metabolismo , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/metabolismo , Esteroides/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxilación , Modelos Moleculares , Mutación , Oxidantes/metabolismo , Oxidación-Reducción , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/aislamiento & purificación , Esteroide Hidroxilasas/metabolismo , Esteroides/química , Especificidad por Sustrato , Testosterona/química , Testosterona/metabolismoRESUMEN
4-Vinyl-1-cyclohexene (VCH) is ovotoxic in B6C3F(1) mice but not in Fischer-344 rats, which can be partially attributed to greater formation of toxic epoxides from VCH in mice compared with rats. Since repeated exposure to VCH is necessary to cause ovotoxicity in mice, it is important to determine whether repeated exposure results in induction of cytochrome P450 (CYP) enzymes involved in its bioactivation. Hepatic microsomes prepared from mice or rats treated repeatedly with VCH demonstrated significantly increased VCH bioactivation in vitro, as assessed by VCH-1,2-epoxide, VCH-7,8-epoxide, or vinylcyclohexene diepoxide (VCD) formation. Mice and rats were then dosed with VCH, VCH-1,2-epoxide, or VCD for 10 days and measured for increases in hepatic microsomal CYP levels or activities. Total hepatic CYP levels were elevated only in microsomes from mice pretreated with VCH or VCH-1,2-epoxide. Immunoblotting analysis of microsomes from VCH-treated rodents revealed elevated levels of CYP2A and CYP2B in mice but not rats. VCH-1,2-epoxide pretreatment also increased CYP2B levels in the mouse. Activities toward specific substrates for CYP2A and CYP2B (coumarin and pentoxyresorufin, respectively) confirmed that VCH and VCH-1,2-epoxide pretreatments resulted in increased catalytic activities of CYP2A and CYP2B in the mouse but not the rat. Pretreatment with phenobarbital, a known inducer of CYP2A and CYP2B, increased VCH bioactivation in both species. Interestingly, metabolism studies with human CYP "Supersomes" reveal that, of eight isoforms tested, only human CYP2E1 and CYP2B6 were capable of significantly catalyzing VCH epoxidation, whereas CYP2B6, CYP2A6, CYP2E1, and CYP3A4 were capable of catalyzing the epoxidation of the monoepoxides.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Ciclohexanos/metabolismo , Animales , Biotransformación , Ciclohexanos/toxicidad , Ciclohexenos , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2E1/fisiología , Sistema Enzimático del Citocromo P-450/fisiología , Inducción Enzimática , Compuestos Epoxi/metabolismo , Femenino , Ratones , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/fisiología , Oxidorreductasas N-Desmetilantes/fisiología , Ratas , Ratas Endogámicas F344 , Especificidad de la EspecieRESUMEN
Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Fenilalanina , Triptófano , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , Escherichia coli , Humanos , Cinética , Hígado/enzimología , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Progesterona/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/fisiología , Mutagénesis Sitio-Dirigida , Animales , Sitios de Unión , Predicción , Humanos , Modelos Moleculares , Estructura Molecular , Polimorfismo Genético , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Isoenzimas/genética , Oxigenasas de Función Mixta/genética , Secuencia de Bases , Citocromo P-450 CYP3A , Cartilla de ADN , Humanos , Mutagénesis , Reacción en Cadena de la PolimerasaRESUMEN
There are conflicting reports about the role of nitric oxide in the down-regulation of cytochrome P450 that occurs when animals or cultured hepatocytes are exposed to inflammatory stimuli. Here, we investigated the participation of NO in the down-regulation of CYP2B1 by bacterial endotoxin (LPS) in rat hepatocytes cultured on Matrigel. LPS caused the down-regulation of CYP2B1 mRNA to 20% of control values within 12 h of treatment, and this was not reversed by concentrations of NO synthase inhibitors that completely blocked NO production. LPS was several orders of magnitude more potent in the down-regulation of CYP2B1 mRNA than in induction of NO production. In contrast, concentrations of LPS in the 1 to 100 ng/ml range induced NO production and produced a rapid down-regulation of CYP2B1 protein to 30% and <5% of control at 6 and 24 h, respectively, that could be completely prevented both by inhibitors of NO synthase and by LY83583, which prevents NO synthase-2 induction. The blockade of CYP2B1 down-regulation by NO synthase inhibitors was reversed by arginine, and the NO donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine mimicked CYP2B1 protein suppression. Taken together, these experiments demonstrate two independent mechanisms of CYP2B1 down-regulation by LPS: a rapid, NO-dependent suppression of the protein occurring at high concentrations of LPS and a slower, NO-independent pretranslational suppression occurring at low concentrations of LPS.
Asunto(s)
Citocromo P-450 CYP2B1/metabolismo , Hepatocitos/enzimología , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , Citocromo P-450 CYP2B1/efectos de los fármacos , Citocromo P-450 CYP2B1/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Lipopolisacáridos/farmacología , Masculino , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B(1) (AFB(1)) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3alpha-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3alpha-hydroxylase and converts AFB(1) predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB(1) regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB(1) detoxification via 3alpha-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB(1) metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB(1) oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11, 265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB(1) 3alpha-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB(1) molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB(1) binding at the active site in either orientation leading to 3alpha- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB(1) 3alpha-hydroxylation and exo-8,9-oxidation.
Asunto(s)
Aflatoxina B1/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutagénesis Sitio-Dirigida/genética , Fenilalanina/metabolismo , Aflatoxina B1/química , Secuencias de Aminoácidos/fisiología , Sitios de Unión/fisiología , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Escherichia coli/genética , Hidroxilación , Inactivación Metabólica/fisiología , Oxigenasas de Función Mixta/química , Modelos Moleculares , Oxidación-Reducción , Fenilalanina/genética , Alineación de Secuencia , Homología de Secuencia , Especificidad por SustratoRESUMEN
Canine cytochromes P450 3A12 and 3A26 are identical in sequence at 481 of 503 amino-acid positions but exhibit different substrate specificities. A recent study utilizing chimeric enzymes and site-directed mutagenesis identified three residues (187, 368, and 369) that contribute to differences in steroid hydroxylation and also indicated the presence of additional determinants of specificity among the 44 carboxyl terminal residues. Therefore, three 3A26 multiple mutants (I187T-S368P-V369I-S467P, I187T-S368P-V369I-S474P, and I187T-S368P-V369I-R476K-I477L-T479A-R480Q) were constructed. Insertion of 3A12 residue Pro-474 into 3A26 I187T-S368P-V369I resulted in metabolite profiles with testosterone, androstenedione, and progesterone very similar to 3A12. Substitution of Pro-474 with Ser in P450 3A12 or human 3A4 significantly increased 2beta-hydroxylase activity with all three steroids. Residue 474 was also found to be an important contributor to diazepam metabolism by the canine and human enzymes. The results provide further evidence for the role of steric constraints exerted by the enzyme in P450 3A-mediated oxidations.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Sustitución de Aminoácidos , Androstenodiona/metabolismo , Animales , Secuencia de Bases , Dominio Catalítico/genética , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN/genética , Diazepam/metabolismo , Perros , Escherichia coli/genética , Humanos , Técnicas In Vitro , Cinética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutagénesis Sitio-Dirigida , Oxidorreductasas N-Desmetilantes/genética , Progesterona/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , ADN Complementario/análisis , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Cartilla de ADN , Escherichia coli , Exones , Humanos , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Peso Molecular , Oxidorreductasas N-Desmetilantes/química , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Citocromo P-450 CYP2B1/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450 , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Esteroide Hidroxilasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Sitios de Unión/genética , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , TransfecciónRESUMEN
The structural basis of cooperativity of progesterone hydroxylation catalyzed by human cytochrome P450 3A4 has been investigated. A recent study suggested that substitution of larger side chains at positions Leu-211 and Asp-214 partially mimics the action of effector by reducing the size of the active site. Based on predictions from molecular modeling that Phe-304 in the highly conserved I helix is involved in both effector and substrate binding, a tryptophan residue was substituted at this position. The purified F304W mutant displayed hyperbolic progesterone hydroxylase kinetics, indicating a lack of homotropic cooperativity. However, the mutant remained responsive to stimulation by alpha-naphthoflavone, exhibiting a 2-fold decrease in the K(m) value for progesterone 6beta-hydroxylation in the presence of 25 microM effector. Combining substitutions to yield the triple mutant L211F/D214E/F304W maintained the V(max) and decreased the K(m) for progesterone 6beta-hydroxylation, minimized stimulation by alpha-naphthoflavone, and decreased the rate of alpha-naphthoflavone oxidation to one-eighth of the wild type. Interestingly, the DeltaA(max) for spectral binding of alpha-naphthoflavone was unaltered in L211F/D214E/F304W. Overall, the results suggest that progesterone and alpha-naphthoflavone are oxidized at separate locations within the P450 3A4 binding pocket, although both substrates appear to have equal access to the reactive oxygen.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Fenilalanina/metabolismo , Benzoflavonas/metabolismo , Sitios de Unión , Clonación Molecular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Humanos , Hidroxilación , Cinética , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Mutación/genética , Mutación/fisiología , Oxidación-Reducción , Fenilalanina/química , Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroide Hidroxilasas/metabolismo , Especificidad por SustratoRESUMEN
The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , ADN Complementario , Humanos , Cetosteroides/metabolismo , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Progesterona/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
The oxidation of a series of seven alkyl ethers of 7-hydroxycoumarin by cytochrome P450 3A4 (CYP3A4) has been studied to probe the active site of the enzyme. TLC of the reaction mixture showed formation of metabolites other than 7-hydroxycoumarin. The separation and characterization of the different metabolites of the C4 to C7 compounds were achieved using a combination of TLC, HPLC, and gas chromatography-electron impact mass spectra. Among the 7-alkoxycoumarins, 7-hexoxycoumarin was found to be the most suitable candidate for investigating the active site of cytochrome CYP3A4, due to the well-separated metabolite peaks on TLC and HPLC. 7-hexoxycoumarin was found to produce three side-chain hydroxylated products besides 7-hydroxycoumarin: 7-(5-hydroxyhexoxy)coumarin, 7-(4-hydroxyhexoxy)coumarin, and 7-(3-hydroxycoumarin). The substitution of residues from substrate recognition sites -1, -4, -5, and -6 of CYP3A4 showed a strong influence on the product profile of 7-hexoxycoumarin, the most prominent effects observed with mutants at residues 119, 301, 305, 370, 373, and 479. The docking of 7-hexoxycoumarin into a molecular model of CYP3A4 also confirmed the presence of these residues within 5 A of the substrate. A comparative study of cytochrome P450 2B1 showed that the active-site mutants F206L, T302V, V363A, and S478G but not V363L exhibited a dramatic decrease in total 7-hexoxycoumarin hydroxylation. The study suggests that although the electronic nature of the substrate is important, enzymatic constraints significantly contribute to CYP3A4 selectivity.
Asunto(s)
Cumarinas/química , Sistema Enzimático del Citocromo P-450/química , Oxigenasas de Función Mixta/química , Sitios de Unión , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Citocromo P-450 CYP2B1/genética , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Colorantes Fluorescentes , Cromatografía de Gases y Espectrometría de Masas , Humanos , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Mutación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-ActividadRESUMEN
CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Oxidorreductasas N-Desmetilantes/genética , Secuencia de Aminoácidos , Androstenodiona/metabolismo , Animales , Secuencia de Bases , Células COS , Células Cultivadas , Embrión de Pollo , Pollos , Clonación Molecular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/química , Inducción Enzimática/efectos de los fármacos , Datos de Secuencia Molecular , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/química , Fenobarbital/farmacología , Filogenia , Progesterona/metabolismo , ARN Mensajero/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Baculoviridae/genética , Sitios de Unión/genética , Citocromo P-450 CYP2B6 , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/química , Activación Enzimática/genética , Humanos , Cinética , Oxidorreductasas N-Desmetilantes/biosíntesis , Oxidorreductasas N-Desmetilantes/química , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Programas Informáticos , Spodoptera/genética , Spodoptera/virología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The structural basis of species differences in cytochrome P450 2B-mediated hydroxylation of 2,2',3,3',6,6'-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli. To facilitate metabolite identification, seven possible products, including three hydroxylated and four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of precursors and Ullmann and crossed Ullmann reactions. HPLC and GC/MS analysis and comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced 4, 5-dihydroxy-236HCB and 5-hydroxy-236HCB, while 2B11 L363V and 2B1 I114V mutants also catalyzed hydroxylation at the 4-position. The amount of products formed by 2B1 mutants I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L was smaller than that of the wild type. I477V exhibited unaltered 236HCB metabolism, and I480V produced twice as much dihydroxy product as the wild type. For 2B11, substitution of Val-114 or Asp-290 with Ile decreased the product yields. Replacement of Leu-363 with Val dramatically altered the profile of 236HCB metabolites. In addition to an increase in the overall level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4-position. Incubation of P450 2B1 with 5-hydroxy-236HCB produced 4,5-dihydroxy-236HCB, which indicates that 4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB. The findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367, 477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Bifenilos Policlorados/metabolismo , Esteroide Hidroxilasas , Hidrocarburo de Aril Hidroxilasas/genética , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B1/genética , Sistema Enzimático del Citocromo P-450/genética , Familia 2 del Citocromo P450 , Escherichia coli/enzimología , Cromatografía de Gases y Espectrometría de Masas , Hidroxilación , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/metabolismoRESUMEN
RPR 106541 (20R-16alpha,17alpha-[butylidenebis(oxy)]-6al pha, 9alpha-difluoro-11beta-hydroxy-17beta-(methylthio)androst a-4-en-3-one) is an airway-selective steroid developed for the treatment of asthma. Two metabolites produced by human liver microsomes were identified as R- and S-sulfoxide diastereomers based on liquid chromatography/mass spectrometry analysis, proton nuclear magnetic resonance, and cochromatography with standards. Sulfoxide formation was determined to be cytochrome P-450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody. Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes. Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer. The high rate of sulfoxidation by CYP3A4 and the blockage of oxidative metabolism at the electronically favored 6beta-position provided advantages for RPR 106541 over other substrates as an active site probe of CYP3A4. Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed. In SRS-4, A305V and F304A showed dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, respectively), but S-diastereomer formation was affected to a lesser extent. A370V (SRS-5) showed decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer. In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A. In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.
Asunto(s)
Androstenos/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Oxigenasas de Función Mixta/antagonistas & inhibidores , Oxigenasas de Función Mixta/genética , Androstenos/química , Animales , Cromatografía Líquida de Alta Presión , Simulación por Computador , Cristalografía por Rayos X , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Perros , Humanos , Espectrometría de Masas , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-Actividad , Sulfóxidos/metabolismoRESUMEN
Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.