RESUMEN
Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Glucosiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Simulación por Computador , Inhibidores Enzimáticos/química , Glucosiltransferasas/química , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Fosfatos de Azúcar/química , Trehalosa/química , Trehalosa/metabolismoRESUMEN
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.
Asunto(s)
Bacillus licheniformis/enzimología , Sacarosa/análogos & derivados , Fosfatos de Azúcar/metabolismo , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Sacarosa/metabolismo , Difracción de Rayos XRESUMEN
The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like α-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give α-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend α-1,4-linked maltooligosaccharides, which are thought to be branched with α-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of α-glucan deposition. A constructed ΔglgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated α-maltose 1-phosphate and maltose but no α-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the ΔglgE mutant on galactose and that of a Δpep2 mutant on maltose were analysed. In both cases, neither accumulation of α-maltose 1-phosphate/α-glucan nor a developmental delay was observed. Thus, high levels of α-maltose 1-phosphate are responsible for the developmental phenotype of the ΔglgE mutant, rather than the lack of α-glucan.
Asunto(s)
Glucanos/metabolismo , Glucosiltransferasas/genética , Esporas Bacterianas/crecimiento & desarrollo , Streptomyces/crecimiento & desarrollo , Fosfatos de Azúcar/metabolismo , Glucógeno/metabolismo , Maltosa/metabolismo , Oligosacáridos/metabolismo , Esporas Bacterianas/genética , Streptomyces/genética , Trehalosa/metabolismoRESUMEN
Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.
Asunto(s)
Nicotiana/enzimología , Liasas de Fósforo-Oxígeno/genética , Proteínas de Plantas/genética , Terpenos/metabolismo , Clonación Molecular , Eritritol/análogos & derivados , Eritritol/biosíntesis , Eritritol/metabolismo , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Redes y Vías Metabólicas , Modelos Moleculares , Liasas de Fósforo-Oxígeno/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Fosfatos de Azúcar/metabolismo , Nicotiana/genéticaRESUMEN
Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.
Asunto(s)
Citrus/microbiología , Trehalosa/fisiología , Factores de Virulencia/metabolismo , Xanthomonas/patogenicidad , Vías Biosintéticas/genética , Citrus/metabolismo , Citrus/fisiología , Resistencia a la Enfermedad , Mutación , Estrés Oxidativo , Fotosíntesis , Enfermedades de las Plantas , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Hojas de la Planta/fisiología , Proteoma , Cloruro de Sodio/metabolismo , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Trehalosa/biosíntesis , Trehalosa/metabolismo , Trehalosa/farmacología , Factores de Virulencia/genética , Xanthomonas/enzimología , Xanthomonas/genéticaRESUMEN
SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins.
Asunto(s)
Cotiledón/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Regulación de la Expresión Génica de las Plantas , Fosfatos de Azúcar/metabolismo , Trehalosa/análogos & derivados , Trehalosa/metabolismoRESUMEN
The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
Asunto(s)
Eritritol/análogos & derivados , Expresión Génica/genética , Hevea/metabolismo , Látex/análisis , Ácido Mevalónico/metabolismo , Goma/metabolismo , Fosfatos de Azúcar/metabolismo , Secuencia de Bases , Carotenoides/metabolismo , Eritritol/metabolismo , Biblioteca de Genes , Genes de Plantas/genética , Hevea/genética , Datos de Secuencia Molecular , ARN de Planta/genética , Análisis de Secuencia de ADN , Terpenos/metabolismo , TranscriptomaRESUMEN
Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.
Asunto(s)
Acetatos/farmacología , Antraquinonas/metabolismo , Ciclopentanos/farmacología , Oxilipinas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rubia/metabolismo , Antraquinonas/análisis , Compuestos de Bifenilo/farmacología , Ciclo del Carbono , Supervivencia Celular , Células Cultivadas , Eritritol/análogos & derivados , Eritritol/metabolismo , Glutamato Deshidrogenasa/efectos de los fármacos , Glutamato Deshidrogenasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Isocitrato Deshidrogenasa/efectos de los fármacos , Isocitrato Deshidrogenasa/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Compuestos Onio/farmacología , Vía de Pentosa Fosfato/efectos de los fármacos , Inmunidad de la Planta , Prolina/análisis , Prolina/efectos de los fármacos , Rubia/citología , Rubia/enzimología , Rubia/crecimiento & desarrollo , Transducción de Señal , Fosfatos de Azúcar/metabolismo , Factores de TiempoRESUMEN
D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.
Asunto(s)
Carotenoides/biosíntesis , Chlorophyta/aislamiento & purificación , Terpenos/metabolismo , California , Células Cultivadas , Chlorophyta/crecimiento & desarrollo , Chlorophyta/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacología , Lovastatina/farmacología , México , Pentosafosfatos/metabolismo , Fosfatos de Azúcar/metabolismo , beta Caroteno/biosíntesisRESUMEN
Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.
Asunto(s)
Eritritol/análogos & derivados , Euphorbiaceae/genética , Euphorbiaceae/metabolismo , Genes de Plantas/genética , Hevea/genética , Goma/metabolismo , Fosfatos de Azúcar/metabolismo , Secuencia de Aminoácidos , Isótopos de Carbono , Clonación Molecular , Bases de Datos Genéticas , Eritritol/metabolismo , Etiquetas de Secuencia Expresada , Regulación de la Expresión Génica de las Plantas , Hevea/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Reacción en Cadena de la Polimerasa , Plantones/genética , Plantones/metabolismo , Coloración y Etiquetado , Xilulosa/análogos & derivados , Xilulosa/metabolismoRESUMEN
In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.
Asunto(s)
Eritritol/análogos & derivados , Eritrocitos/parasitología , Fosfomicina/análogos & derivados , Plasmodium falciparum/efectos de los fármacos , Fosfatos de Azúcar/metabolismo , Animales , Eritritol/metabolismo , Fosfomicina/farmacología , Genes Protozoarios , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The biosynthesis of (2S)-2-methyl-2-(4'-methyl-3'-pentenyl)-8-(3''-methyl-2-butenyl)-2H-1-benzopyran-6-carboxylic acid (gaudichaudianic acid), the major metabolite in leaves and roots of Piper gaudichaudianum Kunth (Piperaceae), has been investigated employing [1-(13)C]-D-glucose as precursor. The labelling pattern in the isolated gaudichaudianic acid was determined by quantitative (13)C NMR spectroscopy analysis and was consistent with involvement of both mevalonic acid and 2-C-methyl-D-erythritol-4-phosphate pathways in the formation of the dimethylallyl- and geranyl-derived moieties. The results confirmed that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for prenylation of p-hydroxybenzoic acid.
Asunto(s)
Benzoatos/metabolismo , Piper/metabolismo , Terpenos/metabolismo , Benzoatos/aislamiento & purificación , Eritritol/análogos & derivados , Eritritol/metabolismo , Glucosa/metabolismo , Hemiterpenos/metabolismo , Ácido Mevalónico/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Compuestos Organofosforados/metabolismo , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Fosfatos de Azúcar/metabolismo , Terpenos/aislamiento & purificaciónRESUMEN
In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.
Asunto(s)
Animales , Eritritol/análogos & derivados , Eritritol/metabolismo , Eritrocitos/parasitología , Fosfomicina/análogos & derivados , Fosfomicina/farmacología , Plasmodium falciparum/metabolismo , Fosfatos de Azúcar/metabolismo , Genes Protozoarios , Reacción en Cadena de la Polimerasa , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrolloRESUMEN
The biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, the two building blocks for isoprenoid biosynthesis, occurs by two independent pathways in plants. The mevalonic pathway operates in the cytoplasm, and the methyl-d-erythritol 4-phosphate (MEP) pathway operates in plastids. Plastidic isoprenoids play essential roles in plant growth and development. Plants must regulate the biosynthesis of isoprenoids to fulfill metabolic requirements in specific tissues and developmental conditions. The regulatory events that modulate the plant MEP pathway are not well understood. In this article, we demonstrate that the CHLOROPLAST BIOGENESIS6 (CLB6) gene, previously shown to be required for chloroplast development, encodes 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase, the last-acting enzyme of the MEP pathway. Comparative analysis of the expression levels of all MEP pathway gene transcripts and proteins in the clb6-1 mutant background revealed that posttranscriptional control modulates the levels of different proteins in this central pathway. Posttranscriptional regulation was also found during seedling development and during fosmidomycin inhibition of the pathway. Our results show that the first enzyme of the pathway, 1-deoxy-d-xylulose 5-phosphate synthase, is feedback regulated in response to the interruption of the flow of metabolites through the MEP pathway.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Eritritol/análogos & derivados , Mutación , Fosfatos de Azúcar/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Proteínas de Cloroplastos , Eritritol/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Datos de Secuencia Molecular , Filogenia , Plantas Modificadas GenéticamenteRESUMEN
Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.
Asunto(s)
Eritritol/análogos & derivados , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Plasmodium falciparum/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfatos de Azúcar/metabolismo , Animales , Antimaláricos/farmacología , Dolicoles/biosíntesis , Eritrocitos/parasitología , Fosfomicina/farmacología , Genes Protozoarios , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Estructura Molecular , Pentosafosfatos/biosíntesis , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Fosfato de Piridoxal/biosíntesis , Espectrometría de Masa por Ionización de Electrospray , Ubiquinona/biosíntesisRESUMEN
Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.
Asunto(s)
Chromobacterium/genética , Indoles/metabolismo , Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Chromobacterium/metabolismo , Indoles/química , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Fosfatos de Azúcar/genética , Fosfatos de Azúcar/metabolismo , Triptófano/biosíntesis , Triptófano/genéticaRESUMEN
Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule
Asunto(s)
Chromobacterium/genética , Indoles/metabolismo , Chromobacterium/metabolismo , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Fosfatos de Azúcar/genética , Fosfatos de Azúcar/metabolismo , Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/genética , Indoles/química , Triptófano/biosíntesis , Triptófano/genéticaRESUMEN
From the stem bark of Bowdichia virgilioides, lupeol, lupeol acetate, sitosterol, stigmasterol and methyl-3-[2-(1-hydroxymethylvinyl)-2,3-dihydrobenzo[b]furan-5-yl]-(E)-2-propenoate have been isolated. The latter is a new compound and it has been named as bowdenol. Their structures were elucidated with the aid of spectroscopic techniques, mostly 1 and 2D NMR. The biogenetic pathway for bowdenol has been suggested.