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 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
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
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
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
Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp(309) in substrate binding and catalysis. These studies predicted an elevated pK(a) for Asp(309) and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp(309) with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp(309) based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK(a) of 8.2 observed for androstenedione binding to the side chain of Asp(309). To our knowledge, this work represents the first experimental assignment of a pK(a) value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7-8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.
Asunto(s)
Aromatasa/química , Ácido Aspártico/química , Mutación , Conformación Proteica , Andrógenos/química , Andrógenos/metabolismo , Aromatasa/metabolismo , Ácido Aspártico/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Estrógenos/química , Estrógenos/metabolismo , Humanos , Oxidación-Reducción , Espectrometría RamanRESUMEN
A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N- and C-terminal domains suggests an evolutionary model involving a plasmid-mediated horizontal gene transfer from the donor Rhodococcus jostiiâ RHA1 to the receiving A. radioresistensâ S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coliâ BL21, together with the A. radioresistensâ Baeyer-Villiger monooxygenase, allowed the recombinant bacteria to grow on long- and medium-chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium-chain alkanes overlapping AlkB and in the first step of sub-terminal oxidation of long-chain alkanes. It was also demonstrated that CYP116B5 is a self-sufficient cytochrome P450 consisting of a heme domain (aa 1-392) involved in the oxidation step of n-alkanes degradation, and its reductase domain (aa 444-758) comprising the NADPH-, FMN- and [2Fe2S]-binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.
Asunto(s)
Acinetobacter/enzimología , Acinetobacter/genética , Alcanos/metabolismo , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Acinetobacter/crecimiento & desarrollo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Evolución Biológica , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Evolución Molecular , Transferencia de Gen Horizontal , Hemo/química , Italia , Datos de Secuencia Molecular , NADP/metabolismo , Oxidación-Reducción , Filogenia , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Alineación de Secuencia , Microbiología del SueloRESUMEN
This paper reports the structure of the double mutant Asp251Gly/Gln307His (named A2) generated by random mutagenesis, able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. The 3D structure of the substrate-free mutant shows a conformation similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on I-helix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules. The impact of these structural features on thermal stability, reduction potential and electron transfer is investigated. The data demonstrate that a single mutation far from the active site triggers an increase in protein flexibility in a key region, shifting the conformational equilibrium toward the closed form that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/ultraestructura , Diclofenaco/química , Ibuprofeno/química , Simulación del Acoplamiento Molecular/métodos , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/ultraestructura , Tolbutamida/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Simulación por Computador , Sistema Enzimático del Citocromo P-450/genética , Activación Enzimática , Datos de Secuencia Molecular , Mutación/genética , NADPH-Ferrihemoproteína Reductasa/genética , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Inhibition of human cytochrome P450 2A6 has been demonstrated to play an important role in nicotine metabolism and consequent smoking habits. Here, the "molecular Lego" approach was used to achieve the first reported electrochemical signal of human CYP2A6 and to improve its catalytic efficiency on electrode surfaces. The enzyme was fused at the genetic level to flavodoxin from Desulfovibrio vulgaris (FLD) to create the chimeric CYP2A6-FLD. Electrochemical characterization by cyclic voltammetry shows clearly defined redox transitions of the haem domain in both CYP2A6 and CYP2A6-FLD. Electrocatalysis experiments using coumarin as substrate followed by fluorimetric quantification of the product were performed with immobilized CYP2A6 and CYP2A6-FLD. Comparison of the kinetic parameters showed that coumarin catalysis was carried out with a higher efficiency by the immobilized CYP2A6-FLD, with a calculated kcat value significantly higher (P < 0.005) than that of CYP2A6, whereas the affinity for the substrate (KM) remained unaltered. The chimeric system was also successfully used to demonstrate the inhibition of the electrochemical activity of the immobilized CYP2A6-FLD, toward both coumarin and nicotine substrates, by tranylcypromine, a potent and selective CYP2A6 inhibitor. This work shows that CYP2A6 turnover efficiency is improved when the protein is linked to the FLD redox module, and this strategy can be utilized for the development of new clinically relevant biotechnological approaches suitable for deciphering the metabolic implications of CYP2A6 polymorphism and for the screening of CYP2A6 substrates and inhibitors.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450/farmacología , Técnicas Electroquímicas/métodos , Prevención del Hábito de Fumar , Secuencia de Bases , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , Cese del Hábito de FumarRESUMEN
Pectin is a renewable, non-toxic and biodegradable polymer made of galacturonic acid units. Its polar groups make it suitable for complexing and supporting metallic nanoparticles (NPs). This work aimed to produce antibacterial nanocomposites using pectin and acoustic cavitation. The metal NPs (Au or Ag) were deposited using ultrasound (US, 21â kHz, 50â W) and compared with those achieved with mechanical stirring. The impact of the reducing agents (NaBH4, ascorbic acid) on the dispersion and morphology of the resulting NPs was also assessed. Characterization by diffuse reflectance (DR) UV-Vis-NIR spectroscopy and field emission scanning electron microscopy (FESEM) showed that the use of US improves the dispersion and decreases the size of both Au and Ag NPs. Moreover, with Au NPs, avoiding external reductants led to smaller NPs and more uniform in size. The prepared NPs were functionalized with oxytetracycline in water and tested against Escherichia coli (gram negative) and Staphylococcus epidermidis (gram positive) via the Kirby-Bauer test. The results show a better antibacterial activity of the functionalized nanoparticles compared to antibiotic-free NPs and pure oxytetracycline, advising the potential of the nanoparticles as drug carriers. These findings underscore the significance of US-assisted synthesis, paving the way to new environmentally friendly antimicrobial materials.
Asunto(s)
Antibacterianos , Escherichia coli , Oro , Nanopartículas del Metal , Pruebas de Sensibilidad Microbiana , Pectinas , Plata , Staphylococcus epidermidis , Pectinas/química , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Plata/química , Oro/química , Escherichia coli/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Staphylococcus epidermidis/efectos de los fármacos , Ondas Ultrasónicas , Tamaño de la PartículaRESUMEN
Cytochrome P450 aromatase (CYP19A1) is the only enzyme known to catalyze the biosynthesis of estrogens from androgens. The crystal structure of human placental aromatase (pArom) has paved the way toward understanding the structure-function relationships of this remarkable enzyme. Using an amino terminus-truncated recombinant human aromatase (rArom) construct, we investigate the roles of key amino acids in the active site, at the intermolecular interface, inside the access channel, and at the lipid-protein boundary for their roles in enzyme function and higher-order organization. Replacing the active site residue D309 with an N yields an inactive enzyme, consistent with its proposed involvement in aromatization. Mutation of R192 at the lipid interface, pivotal to the proton relay network in the access channel, results in the loss of enzyme activity. In addition to the distal catalytic residues, we show that mutation of K440 and Y361 of the heme-proximal region critically interferes with substrate binding, enzyme activity, and heme stability. The D-E loop deletion mutant Del7 that disrupts the intermolecular interaction significantly reduces enzyme activity. However, the less drastic Del4 and point mutants E181A and E181K do not. Furthermore, native gel electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation are used to show that mutations in the intermolecular interface alter the quaternary organization of the enzyme in solution. As a validation for interpretation of the mutational results in the context of the innate molecule, we determine the crystal structure of rArom to show that the active site, tertiary, and quaternary structures are identical to those of pArom.
Asunto(s)
Aromatasa/química , Secuencia de Aminoácidos , Aromatasa/genética , Inhibidores de la Aromatasa/química , Dominio Catalítico , Cristalización , Femenino , Humanos , Cinética , Placenta/enzimología , Embarazo , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Difracción de Rayos XRESUMEN
Aromatase (CYP19) is involved in steroidogenesis, catalyzing the conversion of androgens into estrogens through a unique reaction that causes the aromatization of the A ring of the steroid. The enzyme is widely distributed and well conserved among species as it plays a crucial role in physiological processes such as control of reproduction and neuroprotection. It has also been a subject of intense research both at the biotechnological level in drug development due to its involvement in estrogen-dependent tumors and at a fundamental biochemical level because there are numerous questions regarding its reaction mechanism. This review will report the great progress made in this area.
Asunto(s)
Aromatasa/metabolismo , Biotecnología , Aromatasa/química , Aromatasa/aislamiento & purificación , Estrógenos/biosíntesis , Estrógenos/química , Humanos , Modelos Moleculares , Simulación de Dinámica MolecularRESUMEN
Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2 O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2 O2 . The full-length enzyme (CYP116B5-fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2 O2 (CYP116B5-hd), and sarcosine (CYP116B5-SOX) as source of electrons. CYP116B5-SOX performs better than CYP116B5-fl and CYP116B5-hd showing 10- and 3-folds higher activity, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Ingeniería de Proteínas , Sistema Enzimático del Citocromo P-450/metabolismo , Catálisis , Hemo/química , Hemo/metabolismoRESUMEN
The soluble, catalytically self-sufficient cytochrome P450 BM3 from Bacillus megaterium is a good candidate as biocatalyst for the synthesis of drug metabolites. To this end, error-prone polymerase chain reaction (PCR) was used to generate a library of P450 BM3 mutants with novel activities toward drugs. The double mutant Asp251Gly/Gln307His (A2) with activities towards diclofenac, ibuprofen and tolbutamide was identified by screening with the alkali method. This is based on the detection of NADPH oxidation during enzymatic turnover on whole Escherichia coli cells heterologously expressing the P450 BM3 mutants in the presence of the target substrates. The three drugs screened are marker substrates of human liver cytochromes P450 belonging to the 2C subfamily. Interestingly the mutations Asp251Gly/Gln307His are located on the protein surface and they are not directly involved in substrate binding and turnover. Dissociation constants and K(M) values of mutant A2 for diclofenac, ibuprofen and tolbutamide are in the micromolar range. Catalysis leads to hydroxylations in specific positions, producing 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide, respectively.