RESUMO
The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the â¼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , NADPH-Ferri-Hemoproteína Redutase/química , Multimerização Proteica , Cristalografia por Raios X , Medição da Troca de Deutério , Espectrometria de Massas , Domínios Proteicos , Estrutura Quaternária de ProteínaRESUMO
Extensive use of pyrethroids for malaria control in Africa has led to widespread pyrethroid resistance in the two major African vectors of malaria An. gambiae and An. funestus. This is often associated with constitutively elevated levels of cytochrome P450s involved with pyrethroid metabolism and detoxification. P450s have the capacity to metabolise diverse substrates, which raises concerns about their potential to cause cross-resistance. A bank of seven recombinant P450s from An. gambiae (CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 9J5) and An. funestus (CYP6P9a) commonly associated with pyrethroid resistance were screened against twelve insecticides representing the five major classes of insecticides recommended by WHO for malaria control; permethrin, etofenprox and bifenthrin (type I pyrethroids), deltamethrin, lambda cyhalothrin and cypermethrin (type II pyrethroids), DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos methyl and fenitrothion (organophosphates) and pyriproxyfen (juvenile hormone analogue). DDT was not metabolised by the P450 panel, while bendiocarb was only metabolised by CYP6P3. Pyrethroids and pyriproxyfen were largely susceptible to metabolism by the P450 panel, as were organophosphates, which are activated by P450s. Primiphos-methyl is increasingly used for malaria control. Examination of the pirimiphos-methyl metabolites generated by CYP6P3 revealed both the active pirimiphos-methyl-oxon form and the inactive oxidative cleavage product 2-diethylamino-6-hydroxy-4-methylpyrimidine. The inhibition profile of CYPs 6M2, 6P2, 6P3, 6P9a and 9J5 was also examined using diethoxyfluorescein (DEF) as the probe substrate. Bendiocarb was the weakest inhibitor with IC50â¯>â¯100⯵M across the P450 panel, while CYP6M2 showed strongest inhibition by malathion (IC50 0.7⯵M). The results suggest that P450s present at elevated levels in two major Anopheline vectors of malaria in Africa have the capacity to metabolise a diverse range of pyrethroid and organophosphate insecticides as well as pyriproxyfen that could impact vector control.
Assuntos
Anopheles/efeitos dos fármacos , Anopheles/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Resistência a Inseticidas , Malária/transmissão , Mosquitos Vetores/efeitos dos fármacos , Piretrinas/farmacologia , Animais , Anopheles/classificação , Controle de Mosquitos/métodos , Mosquitos Vetores/parasitologia , Compostos Organotiofosforados/farmacologia , Especificidade da EspécieRESUMO
Flavocytochrome P450 BM3 is a natural fusion protein constructed of cytochrome P450 and NADPH-cytochrome P450 reductase domains. P450 BM3 binds and oxidizes several mid- to long-chain fatty acids, typically hydroxylating these lipids at the ω-1, ω-2 and ω-3 positions. However, protein engineering has led to variants of this enzyme that are able to bind and oxidize diverse compounds, including steroids, terpenes and various human drugs. The wild-type P450 BM3 enzyme binds inefficiently to many azole antifungal drugs. However, we show that the BM3 A82F/F87V double mutant (DM) variant binds substantially tighter to numerous azole drugs than does the wild-type BM3, and that their binding occurs with more extensive heme spectral shifts indicative of complete binding of several azoles to the BM3 DM heme iron. We report here the first crystal structures of P450 BM3 bound to azole antifungal drugs - with the BM3 DM heme domain bound to the imidazole drugs clotrimazole and tioconazole, and to the triazole drugs fluconazole and voriconazole. This is the first report of any protein structure bound to the azole drug tioconazole, as well as the first example of voriconazole heme iron ligation through a pyrimidine nitrogen from its 5-fluoropyrimidine ring.
Assuntos
Antifúngicos/química , Azóis/química , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise Espectral , Relação Estrutura-AtividadeRESUMO
Pyrethroid resistance is widespread in the malaria vector Anopheles gambiae leading to concerns about the future efficacy of bednets with pyrethroids as the sole active ingredient. The incorporation of pyriproxyfen (PPF), a juvenile hormone analogue, into pyrethroid treated bednets is being trialed in Africa. Pyrethroid resistance is commonly associated with elevated levels of P450 expression including CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 6Z2 and 9J5. Having expressed these P450s in E. coli we find all are capable of metabolizing PPF. Inhibition of these P450s by permethrin, deltamethrin and PPF was also examined. Deltamethrin and permethrin were moderate inhibitors (IC50 1-10 µM) of diethoxyfluorescein (DEF) activity for all P450s apart from CYP6Z2 (IC50 > 10 µM), while PPF displayed weaker inhibition of all P450s (IC50 > 10 µM) except CYP's 6Z2 and 6P2 (IC50 1-10 µM). We found evidence of low levels of cross resistance between PPF and other insecticide classes by comparing the efficacy of PPF in inhibiting metamorphosis and inducing female sterility in an insecticide susceptible strain of An. gambiae and a multiple resistant strain from Cote d'Ivoire.
Assuntos
Anopheles/efeitos dos fármacos , Anopheles/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Piridinas/farmacologia , Animais , Côte d'Ivoire , Feminino , Metamorfose Biológica , Organismos Geneticamente Modificados , ReproduçãoRESUMO
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51.
Assuntos
Inibidores de 14-alfa Desmetilase/isolamento & purificação , Antiprotozoários/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorometria/métodos , Esterol 14-Desmetilase/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Inibidores de 14-alfa Desmetilase/farmacologia , Animais , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Descoberta de Drogas/métodos , Fluorescência , Ensaios de Triagem em Larga Escala/métodos , Humanos , Trypanosoma cruzi/enzimologiaRESUMO
Production of drug metabolites is one area where enzymatic conversion has significant advantages over synthetic chemistry. These high value products are complex to synthesize, but are increasingly important in drug safety testing. The vast majority of drugs are metabolized by cytochromes P450 (P450s), with oxidative transformations usually being highly regio- and stereo-selective. The PPIs (proton pump inhibitors) are drugs that are extensively metabolized by human P450s, producing diverse metabolites dependent on the specific substrate. In the present paper we show that single mutations (A82F and F87V) in the biotechnologically important Bacillus megaterium P450 BM3 enzyme cause major alterations in its substrate selectivity such that a set of PPI molecules become good substrates in these point mutants and in the F87V/A82F double mutant. The substrate specificity switch is analysed by drug binding, enzyme kinetics and organic product analysis to confirm new activities, and X-ray crystallography provides a structural basis for the binding of esomeprazole to the F87V/A82F enzyme. These studies confirm that such 'gatekeeper' mutations in P450 BM3 produce major perturbations to its conformation and substrate selectivity, enabling novel P450 BM3 reactions typical of those performed by human P450s. Efficient transformation of several PPI drugs to human-like products by BM3 variants provides new routes to production of these metabolites.
Assuntos
Bacillus megaterium/genética , Proteínas de Bactérias/genética , Sistema Enzimático do Citocromo P-450/genética , NADPH-Ferri-Hemoproteína Redutase/genética , Inibidores da Bomba de Prótons/metabolismo , Bacillus megaterium/enzimologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Esomeprazol/metabolismo , Humanos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Omeprazol/metabolismo , Oxirredução , Especificidade por SubstratoRESUMO
Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such "gatekeeper" mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/química , Sistema Enzimático do Citocromo P-450/química , NADPH-Ferri-Hemoproteína Redutase/química , Omeprazol/química , Inibidores da Bomba de Prótons/química , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Citocromo P-450 CYP2C19 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Hidroxilação/genética , Mutação , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Omeprazol/farmacocinética , Oxirredução , Estrutura Terciária de Proteína , Inibidores da Bomba de Prótons/farmacocinética , Relação Estrutura-AtividadeRESUMO
Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic "shield" from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K(m) for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.
Assuntos
Bacillus megaterium/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , NAD/metabolismo , Bacillus megaterium/genética , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , Heme/metabolismo , Hidroxilação , Mutação , Oxirredução , Multimerização ProteicaRESUMO
The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1'-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.