RESUMEN
Metabolic profiling of new drugs is limited by the difficulty in obtaining sufficient quantities of minor metabolites for definitive structural identification. Biocatalytic methods offer the potential to produce metabolites that are difficult to synthesize by traditional medicinal chemistry. We hypothesized that the regioselectivity of the drug metabolizing cytochrome P450s could be altered by directed evolution to produce minor metabolites of drugs in development. A biocatalyst library was constructed by DNA shuffling of four CYP3A forms. The library contained 11 ± 4 (mean ± SD) recombinations and 1 ± 1 spontaneous mutations per mutant. On expression in Escherichia coli, 96% of mutants showed detectable activity to at least one probe substrate. Using testosterone as a model drug-like substrate, mutants were found that preferentially formed metabolites produced in only trace amounts by parental forms. A single 1.6L batch culture of one such mutant enabled the facile isolation of 0.3mg of the minor metabolite 1ß-hydroxytestosterone and its ab initio structural determination by 1D- and 2D-NMR spectroscopy.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Descubrimiento de Drogas/métodos , Citocromo P-450 CYP3A/genética , Barajamiento de ADN , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Biblioteca de Genes , Hidroxitestosteronas/metabolismo , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Echinacea/química , Microsomas Hepáticos/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Compuestos Epoxi/metabolismo , Humanos , Hidroxilación , Proteínas Recombinantes/metabolismoRESUMEN
Amino terminal sequence modification of cytochrome P450 enzymes is often necessary to achieve expression in bacteria. The aim of this study was to examine the effect of such modifications on membrane integration and P450 activity. Forms that retained substantial N-terminal hydrophobic sequences remained unaffected by treatments to remove peripheral membrane proteins and were released only by detergent. Truncated P450s 2A13, 2C9 (delta 3-20), 2C19 (delta 3-20), 2D6 (DB11) and 2E1 remained principally membrane-bound, but some P450 was found in the soluble fraction and could be partially extracted by alkaline and high salt treatments. The subcellular localization of P450s 2C9 and 2C19 assessed by fluorescence microscopy mirrored the distribution between subcellular fractions. The MALLLAVFL modified forms of P450 2C9 YFP, P450 2C18 YFP and P450 2C19 YFP were found primarily at the periphery of the cells, whereas the truncated forms of P450 2C9 (delta 3-20) YFP and 2C19 (delta 3-20) YFP were observed at the periphery as well as inside the cells. N-terminal variants of P450s 2C9 and 2C19 showed altered kinetics towards form-selective substrates. Rates of diclofenac 4 -hydroxylation by P450 2C9 and luciferin H-EGE metabolism by P450 2C19 were higher for the MALLLAVFL-modified forms compared with the (delta 3-20) truncated forms despite supplementation of truncated form incubations with additional reductase. Thus, N-terminal sequence modifications changed the degree of membrane integration, potentially affecting subcellular localization, interactions with redox partners, and hence enzymatic activity.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Membrana Celular/enzimología , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Humanos , Proteínas Recombinantes/genética , Eliminación de Secuencia , Especificidad por Sustrato/genéticaRESUMEN
The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Preparaciones Farmacéuticas/metabolismo , Catálisis , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/metabolismo , Escherichia coli , Humanos , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Operón , Proteínas Recombinantes/biosíntesis , Especificidad por SustratoRESUMEN
Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.
Asunto(s)
Amidas/metabolismo , Butilaminas/metabolismo , Echinacea/química , Microsomas Hepáticos/metabolismo , Amidas/química , Butilaminas/química , Cromatografía Líquida de Alta Presión , Humanos , Microsomas Hepáticos/efectos de los fármacos , Extractos Vegetales/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Neoplasias de la Mama/genética , Carcinoma de Células Escamosas/genética , Carcinoma/genética , Sistema Enzimático del Citocromo P-450/genética , Neoplasias Pulmonares/genética , Polimorfismo Genético/genética , Adenocarcinoma/enzimología , Adenocarcinoma/epidemiología , Adenocarcinoma/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/epidemiología , Carcinoma/enzimología , Carcinoma/epidemiología , Carcinoma de Células Grandes/enzimología , Carcinoma de Células Grandes/epidemiología , Carcinoma de Células Grandes/genética , Carcinoma de Células Pequeñas/enzimología , Carcinoma de Células Pequeñas/epidemiología , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/epidemiología , Catálisis , Citocromo P-450 CYP1B1 , Sistema Enzimático del Citocromo P-450/metabolismo , Estradiol/metabolismo , Femenino , Frecuencia de los Genes , Variación Genética , Genotipo , Humanos , Incidencia , Japón/epidemiología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/epidemiología , Masculino , Polimorfismo Conformacional Retorcido-Simple , Valores de Referencia , Medición de Riesgo , Esteroide Hidroxilasas/metabolismoRESUMEN
NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.
Asunto(s)
Compuestos de Anilina/metabolismo , Clorzoxazona/metabolismo , Cumarinas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Dimetilnitrosamina/metabolismo , Microsomas Hepáticos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2E1 , Inhibidores Enzimáticos del Citocromo P-450 , Citocromos b5/antagonistas & inhibidores , Humanos , Cinética , NADH NADPH Oxidorreductasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
Adverse reactions to drugs in which an immune mechanism is responsible for toxicity have been described as idiosyncratic. Understanding these toxic effects is important to enable the identification of patients at risk. The specific toxic side effects considered are heparin-induced thrombocytopenia, penicillin-induced haemolytic anaemia, hepatitis as a result of halothane and tienilic acid therapy, quinine- and quinidine-dependent thrombocytopenia, methyldopa-induced haemolytic anaemia and immune-complex disease following administration of hydralazine, procainamide and penicillamine. The molecular mechanisms of immunotoxicity are presented where such information is available although more than one effect may contribute to the observed pattern of toxicity. The initial events leading to antibody production in certain individuals in response to drug therapy are not understood and, in many of the examples described, antibody production occurs in some patients who do not subsequently experience clinical problems. Clinically serious adverse effects involving immune reactions are infrequent, and a range of genetic and environmental circumstances need to be present simultaneously in an individual before toxicity develops. The ability to metabolise a particular drug has been shown to be one major predisposing factor in toxicity; the immunocompetence of the patient is likely to be another. Both of these considerations are subject to genetic and environmental controls, including infection and disease.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Inmunotoxinas/efectos adversos , HumanosRESUMEN
Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Secuencia de Aminoácidos , Catálisis , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes , Saccharomyces cerevisiae , Especificidad por SustratoRESUMEN
The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs.
Asunto(s)
Arilsulfotransferasa/biosíntesis , Arilsulfotransferasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Animales , Arilsulfotransferasa/metabolismo , Células COS/metabolismo , Dopamina/metabolismo , Estabilidad de Enzimas , Humanos , Isoenzimas/metabolismo , Cinética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.
Asunto(s)
Acetiltransferasas/metabolismo , Carcinógenos/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Heterocíclicos/toxicidad , Mutágenos/toxicidad , NADPH-Ferrihemoproteína Reductasa/metabolismo , Salmonella typhimurium/enzimología , Acetiltransferasas/genética , Aminas/toxicidad , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Relación Dosis-Respuesta a Droga , Activación Enzimática , Fluorenos , Humanos , Isoenzimas , Pruebas de Mutagenicidad/métodos , NADPH-Ferrihemoproteína Reductasa/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Sensibilidad y EspecificidadAsunto(s)
Acetaminofén/efectos adversos , Analgésicos no Narcóticos/efectos adversos , Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Acetaminofén/farmacocinética , Analgésicos no Narcóticos/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Inactivación Metabólica , Isoenzimas/genética , Ratones , Ratones NoqueadosAsunto(s)
Metoprolol/farmacocinética , Microsomas Hepáticos/metabolismo , Animales , Fenómenos Químicos , Química Física , Cimetidina/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Microsomas Hepáticos/efectos de los fármacos , RatasRESUMEN
Tamoxifen is a known hepatocarcinogen in rats and is associated with an increased incidence of endometrial cancer in patients. One mechanism for these actions is via bioactivation, where reactive metabolites are generated that are capable of binding to DNA or protein. Several metabolites of tamoxifen have been identified that appear to predispose to adduct formation. These include alpha-hydroxytamoxifen, alpha,4-dihydroxytamoxifen, and alpha-hydroxy-N-desmethyltamoxifen. Previous studies have shown that cytochrome P450 (P450) enzymes play an important role in the biotransformation of tamoxifen. The aim of our work was to determine which P450 enzymes were capable of producing alpha-hydroxylated metabolites from tamoxifen. When tamoxifen (18 or 250 microM) was used as the substrate, P450 3A4, and to a lesser extent, P450 2D6, P450 2B6, P450 3A5, P450 2C9, and P450 2C19 all produced a metabolite with the same HPLC retention time as alpha-hydroxytamoxifen at either substrate concentration tested. This peak was well-separated from 4-hydroxy-N-desmethyltamoxifen, which eluted substantially later under the chromatographic conditions used. No alpha,4-dihydroxytamoxifen was detected in incubations with any of the forms with tamoxifen as substrate. However, when 4-hydroxytamoxifen (100 microM) was used as the substrate, P450 2B6, P450 3A4, P450 3A5, P450 1B1, P450 1A1, and P450 2D6 all produced detectable concentrations of alpha,4-dihydroxytamoxifen. These studies demonstrate that multiple human P450s, including forms found in the endometrium, may generate reactive metabolites in women undergoing tamoxifen therapy, which could subsequently play a role in the development of endometrial cancer.
Asunto(s)
Antineoplásicos Hormonales/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Humanos , Microsomas Hepáticos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
1. Phase I metabolism of drugs is accomplished by the concerted actions of a limited number of cytochrome P450 enzymes with wide but often overlapping substrate specificites. Although metabolism generally accelerates the clearance of drugs, reactive products may also be generated that cause toxic effects. 2. Because individuals vary in the range and levels of different P450 forms, it is useful to be able to determine the specific isoforms involved in a particular metabolic reaction, in order to estimate the extent of variation within a population in the pharmacokinetics of specific drugs. Such studies may also allow predictions to be made regarding the relative susceptibility of different individuals to possible adverse effects associated with drug treatment. 3. Human cytochrome P450 enzymes are now routinely expressed as recombinant proteins in many different systems, including mammalian cell culture, yeast, baculovirus and Escherichia coli. The latter system is particularly useful when large amounts of protein are required for biophysical studies, but can also be adapted to routine examination of pathways of drug metabolism and toxicology. 4. The present review provides an analysis of strategies used for enhancing cytochrome P450 expression in bacteria and for examining the activity of the recombinant proteins. The potential applications of recombinant P450 are discussed, with particular emphasis on investigation of the roles of cytochrome P450 forms in the metabolism and the toxicity of drugs.
Asunto(s)
Bacterias/enzimología , Bacterias/genética , Sistema Enzimático del Citocromo P-450/biosíntesis , Isoenzimas/biosíntesis , Toxicología/métodos , Secuencia de Aminoácidos , Animales , Sistema Enzimático del Citocromo P-450/genética , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Biotecnología/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Biotecnología/tendencias , Citocromo P-450 CYP2A6 , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/metabolismo , Humanos , Carmin de Índigo , Indoles/metabolismo , Oxigenasas de Función Mixta/genética , Modelos Químicos , Plantas Modificadas Genéticamente , Proteínas Recombinantes/metabolismoRESUMEN
1. Phenacetin O-deethylation catalysed by human liver microcomes has been examined over a substrate concentration range of 2.5 to 700 microM using preparations of eight human liver samples. Michaelis-Menten kinetics described phenacetin oxidation satisfactorily in five of these samples; apparent Km values were in the range of 17.7 to 38.4 microM. 2. In the three other livers a single rectangular hyperbolic relationship did not describe the substrate saturation data adequately; analyses in these three cases requiring two classes of catalytic site. The apparent Km value for the higher affinity class of site in these three samples was within the range quoted above, but limitations imposed by assay sensitivity and phenacetin solubility obviated accurate characterization of the lower affinity class. 3. Estimates of Vmax for the high affinity class of site in the eight livers varied eleven-fold and there was no correlation between either Km or Vmax and microsomal cytochrome P-450 specific content, NADPH cytochrome c (P-450) reductase specific activity or ethylmorphine demethylase activity. 4. Propranolol was a potent competitive inhibitor of phenacetin deethylation with apparent Ki values of 2 to 7 microM describing its effect on the higher affinity class of activity. Propranolol was also an inhibitor of the lower affinity phenacetin deethylase identified in three of the livers, however the mechanism of inhibition could not be characterized. 5. These data suggest the possibility that propranolol oxidation may be mediated in part, by one or more human liver cytochrome P-450 species catalyzing phenacetin oxidation.
Asunto(s)
Microsomas Hepáticos/metabolismo , Fenacetina/metabolismo , Propranolol/farmacología , Adulto , Anciano , Sistema Enzimático del Citocromo P-450/metabolismo , Etilmorfina-N-Demetilasa/metabolismo , Femenino , Humanos , Cinética , Masculino , Microsomas Hepáticos/efectos de los fármacos , Persona de Mediana Edad , NADPH-Ferrihemoproteína Reductasa/metabolismoRESUMEN
Bacterial systems have long been of use in toxicology. In addition to providing general models of enzymes and paradigms for biochemistry and molecular biology, they have been adapted to practical genotoxicity assays. More recently, bacteria also have been used in the production of mammalian enzymes of relevance to toxicology. Escherichia coli has been used to express cytochrome P450, NADPH-cytochrome P450 reductase, flavin-containing monooxygenase, glutathione S-transferase, quinone reductase, sulfotransferase, N-acetyltransferase, UDP-glucuronosyl transferase, and epoxide hydrolase enzymes from humans and experimental animals. The expressed enzymes have been utilized in a variety of settings, including coupling with bacterial genotoxicity assays. Another approach has involved expression of mammalian enzymes directly in bacteria for use in genotoxicity systems. Particularly with Salmonella typhimurium. Applications include both the reversion mutagenesis assay and a system using a chimera with an SOS-response indicator and a reporter.
Asunto(s)
Bacterias/enzimología , Sistema Enzimático del Citocromo P-450/genética , Secuencia de Aminoácidos , Animales , Bacterias/genética , Biotransformación , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Immunoblotting , Datos de Secuencia Molecular , Pruebas de Mutagenicidad , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Respuesta SOS en Genética/efectos de los fármacos , Respuesta SOS en Genética/genética , Xenobióticos/toxicidadRESUMEN
Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens. The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli. A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed. Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT). Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered. The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield. Ferric P450 2E1 was isolated in a mixed spin state. The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax. N-Terminal amino acid sequence analysis yielded the expected first 21 residues. The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme.