RESUMEN
The design of drugs from natural products is a re-emerging area due to the need for bioactive compounds. The exploitation of natural products and their derivatives obtained by biocatalysis is in line with the higher attention given today to new sustainable technologies that better preserve the environment (green chemistry). The research field of cytochromes P450 (CYPs) is continuously providing new enzymes and mutants that produce metabolites suitable for late-stage functionalization for new potential drugs. This review provides an overview of the exploitation of CYPs as biocatalysts in drug synthesis. Additionally, recent progress in protein and metabolic engineering is provided to show how these enzymes offer a toolbox that can be combined with other biocatalytic or chemical processes to build new platforms for the green production of new drugs.
Asunto(s)
Productos Biológicos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Preparaciones Farmacéuticas , Biocatálisis , Oxidación-Reducción , Ingeniería de Proteínas , Especificidad por SustratoRESUMEN
Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.
Asunto(s)
Cynara scolymus , Infertilidad Vegetal , Polen , Infertilidad Vegetal/genética , Cynara scolymus/genética , Polen/genética , Genoma de Planta , Genes de PlantasRESUMEN
The use of enzymes to generate hydrogen, instead of using rare metal catalysts, is an exciting area of study in modern biochemistry and biotechnology, as well as biocatalysis driven by sustainable hydrogen. Thus far, the oxygen sensitivity of the fastest hydrogen-producing/exploiting enzymes, [FeFe]hydrogenases, has hindered their practical application, thereby restricting innovations mainly to their [NiFe]-based, albeit slower, counterparts. Recent exploration of the biodiversity of clostridial hydrogen-producing enzymes has yielded the isolation of representatives from a relatively understudied group. These enzymes possess an inherent defense mechanism against oxygen-induced damage. This discovery unveils fresh opportunities for applications such as electrode interfacing, biofuel cells, immobilization, and entrapment for enhanced stability in practical uses. Furthermore, it suggests potential combinations with cascade reactions for CO2 conversion or cofactor regeneration, like NADPH, facilitating product separation in biotechnological processes. This work provides an overview of this new class of biocatalysts, incorporating unpublished protein engineering strategies to further investigate the dynamic mechanism of oxygen protection and to address crucial details remaining elusive such as still unidentified switching hot-spots and their effects. Variants with improved kcat as well as chimeric versions with promising features to attain gain-of-function variants and applications in various biotechnological processes are also presented.
RESUMEN
The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole-dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system-imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole-nitrogen axial coordination and a low-spin heme FeIII. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between gx (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be -60° and -25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated 14N.
Asunto(s)
Compuestos Férricos , Hemo , Hemo/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Compuestos Férricos/química , Protones , Hierro/química , Imidazoles/química , Sistema Enzimático del Citocromo P-450RESUMEN
Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.
Asunto(s)
Malaria , Monocitos , Humanos , Monocitos/metabolismo , Hidroxilación , Cromatografía de Gases y Espectrometría de Masas , Malaria/metabolismo , Terapia de Inmunosupresión , Procesamiento Proteico-Postraduccional , Familia 4 del Citocromo P450/metabolismoRESUMEN
BACKGROUND: Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). Although it has been used for many years, its mechanism of action remains elusive. H295R cells are, in ACC, an essential tool to evaluate drug mechanisms, although they often lead to conflicting results. METHODS: Using different in vitro biomolecular technologies and biochemical/biophysical experiments, we evaluated how the presence of "confounding factors" in culture media and patient sera could reduce the pharmacological effect of mitotane and its metabolites. RESULTS: We discovered that albumin, the most abundant protein in the blood, was able to bind mitotane. This interaction altered the effect of the drug by blocking its biological activity. This blocking effect was independent of the albumin source or methodology used and altered the assessment of drug sensitivity of the cell lines. CONCLUSIONS: In conclusion, we have for the first time demonstrated that albumin does not only act as an inert drug carrier when mitotane or its metabolites are present. Indeed, our experiments clearly indicated that both albumin and human serum were able to suppress the pharmacological effect of mitotane in vitro. These experiments could represent a first step towards the individualization of mitotane treatment in this rare tumor.
Asunto(s)
Neoplasias de la Corteza Suprarrenal , Carcinoma Corticosuprarrenal , Humanos , Neoplasias de la Corteza Suprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Albúminas , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Mitotano/farmacología , Mitotano/uso terapéutico , Mitotano/metabolismoRESUMEN
The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as 'molecular Lego'. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the 'molecular Lego' approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min−1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.
Asunto(s)
Bacillus megaterium , Hemo , Proteínas Bacterianas/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/metabolismo , Humanos , NADPH-Ferrihemoproteína Reductasa/metabolismo , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Human flavin-containing monooxygenase 3 (FMO3) is a membrane-bound, phase I drug metabolizing enzyme. It is highly polymorphic with some of its variants demonstrating differences in rates of turnover of its substrates: xenobiotics including drugs as well as dietary compounds. In order to measure its in vitro activity and compare any differences between the wild type enzyme and its polymorphic variants, we undertook a systematic study using different engineered proteins, heterologously expressed in bacteria, purified and catalytically characterized with 3 different substrates. These included the full-length as well as the more soluble C-terminal truncated versions of the common polymorphic variants (E158K, V257M and E308G) of FMO3 in addition to the full-length and truncated wild-type proteins. In vitro activity assays were performed with benzydamine, tamoxifen and sulindac sulfide, whose products were measured by HPLC. Differences in catalytic properties between the wild-type FMO3 and its common polymorphic variants were similar to those observed with the truncated, more soluble versions of the enzymes. Interestingly, the truncated enzymes were better catalysts than the full-length proteins. The data obtained point to the feasibility of using the more soluble forms of this enzyme for in vitro drug assays as well as future biotechnological applications possibly in high throughput systems such as bioelectrochemical platforms and biosensors.
Asunto(s)
Oxígeno/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Polimorfismo Genético , Humanos , Modelos Moleculares , Oxidación-Reducción , Oxigenasas/química , Conformación ProteicaRESUMEN
The human cytomegalovirus (HCMV) US12 gene family comprises a set of 10 contiguous genes (US12 to US21) with emerging roles in the regulation of virus cell tropism, virion composition, and immunoevasion. Of all of the US12 gene products, pUS21 shows the highest level of identity with two cellular transmembrane BAX inhibitor motif-containing (TMBIM) proteins: Bax inhibitor-1 and Golgi anti-apoptotic protein, both of which are involved in the regulation of cellular Ca2+ homeostasis and adaptive cell responses to stress conditions. Here, we report the US21 protein to be a viral-encoded ion channel that regulates intracellular Ca2+ homeostasis and protects cells against apoptosis. Indeed, we show pUS21 to be a 7TMD protein expressed with late kinetics that accumulates in ER-derived vesicles. Deletion or inactivation of the US21 gene resulted in reduced HCMV growth, even in fibroblasts, due to reduced gene expression. Ratiometric fluorescence imaging assays revealed that expression of pUS21 reduces the Ca2+ content of intracellular ER stores. An increase in cell resistance to intrinsic apoptosis was then observed as an important cytobiological consequence of the pUS21-mediated alteration of intracellular Ca2+ homeostasis. Moreover, a single point mutation in the putative pore of pUS21 impaired the reduction of ER Ca2+ concentration and attenuated the antiapoptotic activity of pUS21wt, supporting a functional link with its ability to manipulate Ca2+ homeostasis. Together, these results suggest pUS21 of HCMV constitutes a TMBIM-derived viroporin that may contribute to HCMV's overall strategy to counteract apoptosis in infected cells.
Asunto(s)
Canales de Calcio/metabolismo , Citomegalovirus/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Secuencia de Aminoácidos , Apoptosis/fisiología , Calcio/metabolismo , Línea Celular , Citomegalovirus/fisiología , Citoplasma/metabolismo , Homeostasis/fisiología , Humanos , Transporte Iónico/fisiología , Proteínas de la Membrana/metabolismo , Porinas/metabolismo , Sustancias Protectoras/metabolismo , Alineación de Secuencia/métodos , Proteínas Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Virión/metabolismo , Replicación Viral/genéticaRESUMEN
Aromatase is the cytochrome P450 enzyme converting androgens into estrogen in the last phase of steroidogenesis. As estrogens are crucial in reproductive biology, aromatase is found in vertebrates and the invertebrates of the genus Branchiostoma, where it carries out the aromatization reaction of the A-ring of androgens that produces estrogens. Here, we investigate the molecular evolution of this unique and highly substrate-selective enzyme by means of structural, sequence alignment, and homology modeling, shedding light on its key role in species conservation. The alignments led to the identification of a core structure that, together with key and unique amino acids located in the active site and the substrate recognition sites, has been well conserved during evolution. Structural analysis shows what their roles are and the reason why they have been preserved. Moreover, the residues involved in the interaction with the redox partner and some phosphorylation sites appeared late during evolution. These data reveal how highly substrate-selective cytochrome P450 has evolved, indicating that the driving forces for evolution have been the optimization of the interaction with the redox partner and the introduction of phosphorylation sites that give the possibility of modulating its activity in a rapid way.
Asunto(s)
Aromatasa/genética , Evolución Molecular , Secuencia de Aminoácidos , Animales , Aromatasa/química , Aromatasa/metabolismo , Dominio Catalítico , Estrógenos/metabolismo , Humanos , Modelos Moleculares , Alineación de Secuencia , Relación Estructura-Actividad , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
BACKGROUND: In the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites. RESULTS: The effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated. CONCLUSIONS: The study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.
Asunto(s)
Escherichia coli/metabolismo , Hipoglucemiantes/metabolismo , Oxigenasas/metabolismo , Clomifeno/metabolismo , Dasatinib/metabolismo , Escherichia coli/genética , Humanos , Microorganismos Modificados Genéticamente/metabolismo , Oxigenasas/genética , Piperazinas/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMEN
Dye-decolorizing peroxidases (DyP) were originally discovered in fungi for their ability to decolorize several different industrial dyes. DyPs catalyze the oxidation of a variety of substrates such as phenolic and nonphenolic aromatic compounds. Catalysis occurs in the active site or on the surface of the enzyme depending on the size of the substrate and on the existence of radical transfer pathways available in the enzyme. DyPs show the typical features of heme-containing enzymes with a Soret peak at 404-408 nm. They bind hydrogen peroxide that leads to the formation of the so-called Compound I, the key intermediate for catalysis. This then decays into Compound II yielding back Fe(III) at its resting state. Each catalytic cycle uses two electrons from suitable electron donors and generates two product molecules. DyPs are classified as a separate class of peroxidases. As all peroxidases they encompass a conserved histidine that acts as the fifth heme ligand, however all primary DyP sequences contain a conserved GxxDG motif and a distal arginine that is their characteristic. Given their ability to attack monomeric and dimeric lignin model compounds as well as polymeric lignocellulose, DyPs are a promising class of biocatalysts for lignin degradation that not only represents a source of valuable fine chemicals, but it also constitutes a fundamental step in biofuels production. Research efforts are envisioned for the improvement of the activity of DyPs against lignin, through directed evolution, ration protein design, or one-pot combination with other enzymes to reach satisfactory conversion levels for industrial applications.
Asunto(s)
Bacterias/enzimología , Colorantes/metabolismo , Hongos/enzimología , Lignina/metabolismo , Peroxidasas/metabolismo , Bacterias/metabolismo , Biocatálisis , Biocombustibles/análisis , Biocombustibles/microbiología , Biotecnología/métodos , Dominio Catalítico , Colorantes/química , Hongos/metabolismo , Lignina/química , Modelos Moleculares , Peroxidasas/químicaRESUMEN
Inhibition of cytochrome P450 (CYP)-mediated drug metabolism by dietary substances is the main cause of drug-food interactions in humans. The present study reports on the in vitro inhibition assays of human CYP3A4 genetically linked to the reductase domain of bacterial BM3 of Bacillus megaterium (BMR) resulting in the chimeric protein CYP3A4-BMR. The activity of this chimeric enzyme was initially measured colorimetrically with erythromycin as the substrate where KM values similar to published data were determined. Subsequently, the inhibition assays with three different dietary products, grapefruit juice, curcumin, and resveratrol, were carried out with the chimeric enzyme both in solution and immobilized on electrode surfaces. For the solution studies, nicotinamide adenine dinucleotide phosphate was added as the electron donor, whereas the need for this cofactor was obviated in the immobilized enzyme as it was supplied by the electrode. Inhibition of the N-demethylation of erythromycin by CYP3A4-BMR chimera was measured at increasing concentrations of the different dietary compounds with calculated IC50 values of 0.5%, 31 µM, and 250 µM for grapefruit juice, curcumin, and resveratrol measured in solution compared with 0.7%, 24 µM, and 208 µM measured electrochemically, respectively. These data demonstrate the feasibility of the use of both CYP3A4-BMR chimera as well as bioelectrochemistry for in vitro studies of not only drug-food interactions but also prediction of adverse drug reactions in this important P450 enzyme.
Asunto(s)
Curcumina/química , Citocromo P-450 CYP3A/química , Interacciones Alimento-Droga , Jugos de Frutas y Vegetales , Proteínas Recombinantes de Fusión/química , Resveratrol/química , Bacillus megaterium/genética , Citocromo P-450 CYP3A/genética , Humanos , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Human aromatase is the cytochrome P450 catalysing the conversion of androgens into estrogens playing a key role in the endocrine system. Due to this role, it is likely to be a target of the so-called endocrine disrupting chemicals, a series of compounds able to interfere with the hormone system with toxic effects. If on one side the toxicity of some compounds such as bisphenol A is well known, on the other side the toxic concentrations of such compounds as well as the effect of the many other molecules that are in contact with us in everyday life still need a deep investigation. The availability of biological assays able to detect the interaction of chemicals with key molecular targets of the endocrine system represents a possible solution to identify potential endocrine disrupting chemicals. Here the so-called alkali assay previously developed in our laboratory is applied to test the effect of different compounds on the activity of human aromatase. The assay is based on the detection of the alkali product that forms upon strong alkali treatment of the NADP+ released upon enzyme turnover. Here it is applied on human aromatase and validated using anastrozole and sildenafil as known aromatase inhibitors. Out of the small library of compounds tested, resveratrol and ketoconazole resulted to inhibit aromatase activity, while bisphenol A and nicotine were found to exert an inhibitory effect at relatively high concentrations (100µM), and other molecules such as lindane and four plasticizers did not show any significant effect. These data are confirmed by quantification of the product estrone in the same reaction mixtures through ELISA. Overall, the results show that the alkali assay is suitable to screen for molecules that interfere with aromatase activity. As a consequence it can also be applied to other molecular targets of EDCs that use NAD(P)H for catalysis in a high throughput format for the fast screening of many different compounds as endocrine disrupting chemicals. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
Asunto(s)
Inhibidores de la Aromatasa/química , Aromatasa/química , Bioensayo , Disruptores Endocrinos/química , Anastrozol , Aromatasa/genética , Inhibidores de la Aromatasa/análisis , Compuestos de Bencidrilo/análisis , Compuestos de Bencidrilo/química , Disruptores Endocrinos/análisis , Ensayo de Inmunoadsorción Enzimática , Estrona/química , Expresión Génica , Humanos , Cetoconazol/análisis , Cetoconazol/química , Ligandos , NADP/química , Nicotina/análisis , Nicotina/química , Nitrilos/análisis , Nitrilos/química , Fenoles/análisis , Fenoles/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resveratrol , Citrato de Sildenafil/análisis , Citrato de Sildenafil/química , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Estilbenos/análisis , Estilbenos/química , Triazoles/análisis , Triazoles/químicaRESUMEN
Chimerogenesis involving cytochromes P450 is a successful approach to generate catalytically self-sufficient enzymes. However, the connection between the different functional modules should allow a certain degree of flexibility in order to obtain functional and catalytically efficient proteins. We previously applied the molecular Lego approach to develop a chimeric P450 3A4 enzyme linked to the reductase domain of P450 BM3 (BMR). Three constructs were designed with the connecting loop containing no glycine, 3 glycine or 5 glycine residues and showed a different catalytic activity and coupling efficiency. Here we investigate how the linker affects the ability of P450 3A4 to bind substrates and inhibitors. We measure the electron transfer rates and the catalytic properties of the enzyme also in the presence of ketoconazole as inhibitor. The data show that the construct 3A4-5GLY-BMR with the longest loop better retains the binding ability and cooperativity for testosterone, compared to P450 3A4. In both 3A4-3GLY-BMR and 3A4-5GLY-BMR, the substrate induces an increase in the first electron transfer rate and a shorter lag phase related to a domain rearrangements, when compared to the construct without Gly. These data are consistent with docking results and secondary structure predictions showing a propensity to form helical structures in the loop of the 3A4-BMR and 3A4-3GLY-BMR. All three chimeras retain the ability to bind the inhibitor ketoconazole and show an IC50 comparable with those reported for the wild type protein. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
Asunto(s)
Bacillus megaterium/genética , Proteínas Bacterianas/química , Inhibidores del Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/química , Cetoconazol/química , NADPH-Ferrihemoproteína Reductasa/química , Proteínas Recombinantes de Fusión/química , Bacillus megaterium/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Expresión Génica , Humanos , Cetoconazol/metabolismo , Cinética , Ligandos , Simulación del Acoplamiento Molecular , NADPH-Ferrihemoproteína Reductasa/antagonistas & inhibidores , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Testosterona/química , Testosterona/metabolismoRESUMEN
Aromatase catalyzes the biosynthesis of estrogens from androgens. Owing to the physiological importance of this conversion of lipophilic substrates, the interaction with the lipid bilayer for this cytochrome P450 is crucial for its dynamics that must allow an easy access to substrates and inhibitors. Here, the aromatase-anastrozole interaction is studied by combining computational methods to identify possible access/egress routes with the protein inserted in the membrane and experimental tools aimed at the investigation of the effect of the inhibitor on the protein conformation. By means of molecular dynamics simulations of the protein inserted in the membrane, two channels, not detected in the starting crystal structure, are found after a 20-nSec simulation. Trypsin digestion on the recombinant protein shows that the enzyme is strongly protected by the presence of the substrate and even more by the inhibitor. DSC experiments show an increase in the melting temperature of the protein in complex with the substrate (49.3 °C) and the inhibitor (58.7 °C) compared to the ligand-free enzyme (45.9 °C), consistent with a decrease of flexibility of the protein. The inhibitor anastrozole enters the active site of the protein through a channel different from that used from the substrate and promotes a conformational change that stiffens the protein conformation and decreases the protein-protein interaction between different aromatase molecules.
Asunto(s)
Aromatasa/química , Simulación de Dinámica Molecular , Nitrilos/química , Triazoles/química , Anastrozol , Aromatasa/metabolismo , Humanos , Ligandos , Nitrilos/metabolismo , Estructura Cuaternaria de Proteína , Triazoles/metabolismoRESUMEN
BACKGROUND: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic. METHODS: The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. Kd values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292. RESULTS: Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N- and S-oxidation reactions. CONCLUSIONS: Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation. GENERAL SIGNIFICANCE: A single mutation converts Ar-BVMO to a unique S- or N-monooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.
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Acinetobacter/enzimología , Proteínas Bacterianas/química , Etionamida/química , Flavinas/química , Cetonas/química , Oxigenasas de Función Mixta/química , Microbiología del Suelo , Acinetobacter/genética , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos , Arginina/química , Arginina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Etionamida/metabolismo , Flavinas/metabolismo , Expresión Génica , Glicina/química , Glicina/metabolismo , Cetonas/metabolismo , Cinética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid causes significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.
Asunto(s)
Clostridium acetobutylicum/enzimología , Espectroscopía de Resonancia por Spin del Electrón/métodos , Hidrogenasas/química , Proteínas Hierro-Azufre/metabolismo , Mutación , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Dominio Catalítico , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/química , Modelos MolecularesRESUMEN
Human hepatic flavin-containing monooxygenase 3 is a phase I drug-metabolizing enzyme that is responsible for the oxidation of a variety of drugs and xenobiotics. This work reports on a high throughput rapid colorimetric assay for the screening of substrates or inhibitors of this enzyme. The method is based on the competition of two substrates for access to the active site of hFMO3 whereby the enzymatic product of the first drug converts nitro-5-thiobenzoate (TNB, yellow) to 5,5'-dithiobis (2-nitrobenzoate) (DTNB, colourless). Upon addition of a competing substrate, the amount of detected DNTB is decreased. The assay is validated testing three known substrates of hFMO3, namely benzydamine, tozasertib and tamoxifen. The latter drugs resulted in 41%-55% inhibition. In addition, two other drugs also classified as doping drugs, selegiline and clomiphene, were selected based on their chemical structure similarity to known substrates of hFMO3. These drugs showed 21% and 60% inhibition in the colorimetric assay and therefore were proven to be hFMO3 substrates. LC-MS was used to confirm their N-oxide products. Further characterisation of these newly identified hFMO3 substrates was performed determining their Km and kcat values that resulted to be 314 µM and 1.4 min-1 for selegiline and, 18 µM and 0.1 min-1 for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen-containing compounds as substrates/inhibitors of hFMO3.
Asunto(s)
Benzoatos/química , Bencidamina/química , Ácido Ditionitrobenzoico/química , Oxigenasas/química , Piperazinas/química , Polietilenglicoles/química , Tamoxifeno/química , Colorimetría/métodos , HumanosRESUMEN
The newly isolated Clostridium beijerinckii [FeFe]-hydrogenase CbA5H was characterized by Fourier transform infrared spectroscopy coupled to enzymatic activity assays. This showed for the first time that in this enzyme the oxygen-sensitive active state Hox can be simply and reversibly converted to the oxygen-stable inactive Hinact state. This suggests that oxygen sensitivity is not an intrinsic feature of the catalytic center of [FeFe]-hydrogenases (H-cluster), opening new challenging perspectives on the oxygen sensitivity mechanism as well as new possibilities for exploitation in industrial applications.