RESUMEN
The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 µM a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naïve erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches.
RESUMEN
The evolution of resistance by the malaria parasite to artemisinin, the key component of the combination therapy strategies that are at the core of current antimalarial treatments, calls for the urgent identification of new fast-acting antimalarials. The apicoplast organelle is a preferred target of antimalarial drugs because it contains biochemical processes absent from the human host. Fosmidomycin is the only drug in clinical trials targeting the apicoplast, where it inhibits the methyl erythritol phosphate (MEP) pathway. Here, we characterized the antiplasmodial activity of domiphen bromide (DB), another MEP pathway inhibitor with a rapid mode of action that arrests the in vitro growth of Plasmodium falciparum at the early trophozoite stage. Metabolomic analysis of the MEP pathway and Krebs cycle intermediates in 20 µM DB-treated parasites suggested a rapid activation of glycolysis with a concomitant decrease in mitochondrial activity, consistent with a rapid killing of the pathogen. These results present DB as a model compound for the development of new, potentially interesting drugs for future antimalarial combination therapies.
RESUMEN
BACKGROUND: The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans. RESULTS: This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli. GENERAL SIGNIFICANCE: Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities. CONCLUSION: The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Infecciones por Helicobacter/enzimología , Helicobacter pylori/enzimología , Liasas de Fósforo-Oxígeno/metabolismo , Antibacterianos/química , Antibacterianos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Clonación Molecular , Diseño de Fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Eritritol/genética , Prueba de Complementación Genética , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/genética , Helicobacter pylori/genética , Liasas de Fósforo-Oxígeno/antagonistas & inhibidores , Liasas de Fósforo-Oxígeno/genética , Terpenos/metabolismoRESUMEN
The methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore-directed virtual screening and a protein-protein interaction (PPI)-mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4-(cytidine 5-diphospho)-2C-methyl-D-erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water-mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H-furo[3,2-g]chromen-7-one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology.
Asunto(s)
Diseño de Fármacos , Proteínas de Escherichia coli/metabolismo , Ligandos , Imitación Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas de Escherichia coli/genética , Conformación Molecular , Simulación de Dinámica Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Solventes/química , Resonancia por Plasmón de SuperficieRESUMEN
An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of k(off) = 6.1 × 10(-4) ± 7.5 × 10(-3) and 1.3 × 10(-2) ± 1.0 × 10(-2) s(-1), and reaction lengths of x(ß) = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.
Asunto(s)
Antibacterianos/análisis , Antimaláricos/análisis , Técnicas Biosensibles/instrumentación , Descubrimiento de Drogas , Nanotecnología/instrumentación , Análisis Espectral/instrumentación , Antibacterianos/química , Antimaláricos/química , Técnicas Biosensibles/métodos , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Enzimas Inmovilizadas , Escherichia coli/genética , Humanos , Estructura Molecular , Nanotecnología/métodos , Sensibilidad y Especificidad , Análisis Espectral/métodos , Transferasas/química , Transferasas/genética , Transferasas/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: The present paper aims to study the inhibition of Candida albicans growth as candidiasis treatment, using seeds of Lepidium sativum as source. METHODS: In vitro assays were carried out on the antifungal activity of three kinds of extracts from L. sativum seeds against four strains of C. albicans, then testing the same phytochemicals on the inhibition of Lipase (LCR). A new in silico study was achieved using molecular docking, with Autodock vina program, to find binding affinity of two important and major lepidine alkaloids (lepidine E and B) towards the four enzymes secreted by C. albicans as target drugs, responsible of vitality and virulence of this yeast cells: Lipase, Serine/threonine phosphatase, Phosphomannose isomerase and Sterol 14-alpha demethylase (CYP51). RESULTS: The results of the microdillution assay show that the hexanic and alkaloidal extracts have an antifungal activity with MICs: 2.25 mg/ml and 4.5mg/ml, respectively. However, Candida rugosa lipase assay gives a remarkable IC50 values for the hexanic extract (1.42± 0.04 mg/ml) followed by 1.7± 0.1 and 2.29 ± 0.09 mg/ml of ethyl acetate and alkaloidal extracts respectively. The molecular docking confirms a significant correlation between C. albicans growth and inhibition of crucial enzymes involved in the invasion mechanism and cellular metabolisms, for the first time there were an interesting and new positive results on binding modes of lepidine E and B on the four studied enzymes. CONCLUSION: Through this work, we propose Lepidine B & E as potent antifungal drugs.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Lepidium sativum , Simulación del Acoplamiento Molecular , Extractos Vegetales/farmacología , Semillas , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Candida albicans/enzimología , Candida albicans/crecimiento & desarrollo , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Lepidium sativum/química , Lipasa/antagonistas & inhibidores , Lipasa/metabolismo , Manosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Manosa-6-Fosfato Isomerasa/metabolismo , Terapia Molecular Dirigida , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Conformación Proteica , Semillas/química , Relación Estructura-Actividad , VirulenciaRESUMEN
Combination therapies, where two drugs acting through different mechanisms are administered simultaneously, are one of the most efficient approaches currently used to treat malaria infections. However, the different pharmacokinetic profiles often exhibited by the combined drugs tend to decrease treatment efficacy as the compounds are usually eliminated from the circulation at different rates. To circumvent this obstacle, we have engineered an immunoliposomal nanovector encapsulating hydrophilic and lipophilic compounds in its lumen and lipid bilayer, respectively. The antimalarial domiphen bromide has been encapsulated in the liposome membrane with good efficiency, although its high IC50 of ca. 1 µM for living parasites complicates its use as immunoliposomal therapy due to erythrocyte agglutination. The conjugation of antibodies against glycophorin A targeted the nanocarriers to Plasmodium-infected red blood cells and to gametocytes, the sole malaria parasite stage responsible for the transmission from the human to the mosquito vector. The antimalarials pyronaridine and atovaquone, which block the development of gametocytes, have been co-encapsulated in glycophorin A-targeted immunoliposomes. The co-immunoliposomized drugs have activities significantly higher than their free forms when tested in in vitro Plasmodium falciparum cultures: Pyronaridine and atovaquone concentrations that, when encapsulated in immunoliposomes, resulted in a 50% inhibition of parasite growth had no effect on the viability of the pathogen when used as free drugs.
RESUMEN
The X-ray crystal structure of the 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS) from Arabidopsis thaliana has been solved at 2.3 A resolution in complex with a cytidine-5-monophosphate (CMP) molecule. This is the first structure determined of an MCS enzyme from a plant. Major differences between the A. thaliana and bacterial MCS structures are found in the large molecular cavity that forms between subunits and involve residues that are highly conserved among plants. In some bacterial enzymes, the corresponding cavity has been shown to be an isoprenoid diphosphate-like binding pocket, with a proposed feedback-regulatory role. Instead, in the structure from A. thaliana the cavity is unsuited for binding a diphosphate moiety, which suggests a different regulatory mechanism of MCS enzymes between bacteria and plants.
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Arabidopsis/enzimología , Bacterias/enzimología , Enzimas/química , Eritritol/análogos & derivados , Terpenos/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Bacterias/genética , Sitios de Unión , Cristalografía por Rayos X , Enzimas/aislamiento & purificación , Eritritol/biosíntesis , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
We have developed a new liquid chromatography-electrospray ionization tandem mass spectrometry methodology based on 2-picolylamine derivatization and positive ion mode detection for abscisic acid (ABA) identification. The selected reaction leads to the formation of an amide derivative which contains a highly active pyridyl group. The enhanced ionization allows for a 700-fold increase over commonly monitored unmodified ABA, which in turn leads to excellent limits of detection and quantification values of 0.03 and 0.15ngmL-1, respectively. This method has been validated in the highly complex matrix of a red blood cell extract. In spite of the high sensitivity achieved, ABA could not be detected in Plasmodium falciparum-infected red blood cells, suggesting that, if present, it will be found either in ultratrace amounts or as brief bursts at defined time points within the intraerythrocytic cycle and/or in the form of a biosynthetic analogue.
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Ácido Abscísico/análisis , Eritrocitos/metabolismo , Malaria Falciparum/metabolismo , Ácidos Picolínicos/química , Plasmodium falciparum/metabolismo , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Reguladores del Crecimiento de las Plantas/análisisRESUMEN
A practical experiment on the PCR is described that has been used over several years as part of an undergraduate biochemistry and molecular biology course for chemistry students. In the first experimental session, students prepare their own DNA samples from epithelial cells of the mouth and use them as templates in the PCR. In the second session, they analyze the amplified DNA by electrophoresis and determine their Rh factor.
RESUMEN
Plastid isoprenoids are synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE. In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene. The plant polypeptide, GCPE, contains two structural domains that are absent in the E. coli protein: an N-terminal extension and a central domain of 30 kDa. We demonstrate that the N-terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis. Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE-defective E. coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Plastidios/metabolismo , Terpenos/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Genes Reporteros , Prueba de Complementación Genética , Datos de Secuencia Molecular , Oxidorreductasas , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Evolución Molecular , Genes de Plantas , Transferasas/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , ADN Complementario/genética , ADN Complementario/metabolismo , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Modelos Genéticos , Datos de Secuencia Molecular , Pentosafosfatos/metabolismo , Fenotipo , Filogenia , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plastidios/enzimología , Plastidios/genética , Transferasas/metabolismo , Transformación GenéticaRESUMEN
Tuberculosis is one of the leading infectious diseases in humans. Discovering new treatments for this disease is urgently required, especially in view of the emergence of multiple drug resistant organisms and to reduce the total duration of current treatments. The synthesis of isoprenoids in Mycobacterium tuberculosis has been reported as an interesting pathway to target, and particular attention has been focused on the methylerythritol phosphate (MEP) pathway comprising the early steps of isoprenoid biosynthesis. In this context we have studied the enzyme 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase (CMS), the third enzyme in the MEP pathway, since the lack of a resolved structure of this protein in M. tuberculosis has seriously limited its use as a drug target. We performed homology modeling of M. tuberculosis CMS in order to provide a reliable model for use in structure-based drug design. After evaluating the quality of the model, we performed a thorough study of the catalytic site and the dimerization interface of the model, which suggested the most important sites (conserved and non-conserved) that could be useful for drug discovery and mutagenesis studies. We found that the metal coordination of CDP-methylerythritol in M. tuberculosis CMS differs substantially with respect to the Escherichia coli variant, consistent with the fact that the former is able to utilize several metal ions for catalysis. Moreover, we propose that electrostatic interactions could explain the higher affinity of the MEP substrate compared with the cytosine 5'-triphosphate substrate in the M. tuberculosis enzyme as reported previously.
Asunto(s)
Proteínas Bacterianas/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Nucleotidiltransferasas/química , Terpenos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Vías Biosintéticas , Cristalografía por Rayos X , Citidina Difosfato/química , Citidina Difosfato/metabolismo , Eritritol/análogos & derivados , Eritritol/biosíntesis , Eritritol/química , Enlace de Hidrógeno , Metales/química , Metales/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Mycobacterium tuberculosis/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Fosfatos de Azúcar/biosíntesis , Fosfatos de Azúcar/química , TermodinámicaRESUMEN
The 2C-methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl pyrophosphate and its isomer dimethylallyl pyrophosphate, which are the precursors of isoprenoids, is present in plants, in the malaria parasite Plasmodium falciparum and in most eubacteria, including pathogenic agents. However, the MEP pathway is absent from fungi and animals, which have exclusively the mevalonic acid pathway. Given the characteristics of the MEP pathway, its enzymes represent potential targets for the generation of selective antibacterial, antimalarial and herbicidal molecules. We have focussed on the enzyme 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase (CMK), which catalyses the fourth reaction step of the MEP pathway. A molecular dynamics simulation was carried out on the CMK dimer complex, and protein-protein interactions analysed, considering also water-mediated interactions between monomers. In order to find small molecules that bind to CMK and disrupt dimer formation, interactions observed in the dynamics trajectory were used to model a pharmacophore used in database searches. Using an intensity-fading matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry approach, one compound was found to interact with CMK. The data presented here indicate that a virtual screening approach can be used to identify candidate molecules that disrupt the CMK-CMK complex. This strategy can contribute to speeding up the discovery of new antimalarial, antibacterial, and herbicidal compounds.
Asunto(s)
Proteínas de Escherichia coli/química , Fosfotransferasas/química , Multimerización de Proteína , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Solventes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad EstáticaRESUMEN
A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.
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Eritritol/análogos & derivados , Eritritol/análisis , Proteínas de Escherichia coli/análisis , Nucleotidiltransferasas/análisis , Colorimetría/métodos , Eritritol/biosíntesis , Eritritol/metabolismo , Proteínas de Escherichia coli/metabolismo , Pirofosfatasa Inorgánica , Nucleotidiltransferasas/metabolismo , Fosfatos , Colorantes de Rosanilina , Análisis EspectralRESUMEN
We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.
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Proteínas de Escherichia coli/análisis , Escherichia coli/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Espectrofotometría/métodos , Adenosina Difosfato/análisis , Automatización , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/aislamiento & purificación , Cinética , L-Lactato Deshidrogenasa/metabolismo , Microquímica , NAD/metabolismo , Fosfoenolpiruvato/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato ( Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe ( r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.
Asunto(s)
Carotenoides/biosíntesis , Biología Computacional/métodos , Enzimas/genética , Frutas/genética , Solanum lycopersicum/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Enzimas/metabolismo , Etiquetas de Secuencia Expresada , Frutas/enzimología , Frutas/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/enzimología , Solanum lycopersicum/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.