RESUMEN
This work describes the reactivity and properties of fluorinated derivatives (F-PD and F-PDO) of plasmodione (PD) and its metabolite, the plasmodione oxide (PDO). Introduction of a fluorine atom on the 2-methyl group markedly alters the redox properties of the 1,4-naphthoquinone electrophore, making the compound highly oxidizing and particularly photoreactive. A fruitful set of analytical methods (electrochemistry, absorption and emission spectrophotometry, and HRMS-ESI) have been used to highlight the products resulting from UV photoirradiation in the absence or presence of selected nucleophiles. With F-PDO and in the absence of nucleophile, photoreduction generates a highly reactive ortho-quinone methide (o-QM) capable of leading to the formation of a homodimer. In the presence of thiol nucleophiles such as ß-mercaptoethanol, which was used as a model, o-QMs are continuously regenerated in sequential photoredox reactions generating mono- or disulfanylation products as well as various unreported sulfanyl products. Besides, these photoreduced adducts derived from F-PDO are characterized by a bright yellowish emission due to an excited-state intramolecular proton transfer (ESIPT) process between the dihydronapthoquinone and benzoyl units. In order to evidence the possibility of an intramolecular coupling of the o-QM intermediate, a synthetic route to the corresponding anthrones is described. Tautomerization of the targeted anthrones occurs and affords highly fluorescent stable hydroxyl-anthraquinones. Although probable to explain the intense visible fluorescence emission also observed in tobacco BY-2 cells used as a cellular model, these coupling products have never been observed during the photochemical reactions performed in this study. Our data suggest that the observed ESIPT-induced fluorescence most likely corresponds to the generation of alkylated products through reduction species, as demonstrated with the ß-mercaptoethanol model. In conclusion, F-PDO thus acts as a novel (pro)-fluorescent probe for monitoring redox processes and protein alkylation in living cells.
Asunto(s)
Indolquinonas , Vitamina K 3/análogos & derivados , Mercaptoetanol , Indolquinonas/química , AntraquinonasRESUMEN
In plants, geranylgeranyl diphosphate (GGPP, C20 ) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10 )-indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate (MeJA), and was similar to the gene encoding type-I protein geranylgeranyltransferase_ß subunit (CrPGGT-I_ß), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co-localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus-induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2-vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic-overexpression of CrGGPPS2 had no effect on the main GGPP-derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT-I_ß, similar to CrGGPPS2-vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis.
Asunto(s)
Catharanthus/enzimología , Farnesiltransferasa/metabolismo , Monoterpenos/metabolismo , Proteínas de Plantas/metabolismo , Alcaloides de Triptamina Secologanina/metabolismo , Catharanthus/genética , Catharanthus/metabolismo , Farnesiltransferasa/genética , Redes y Vías Metabólicas , Filogenia , Plastidios/metabolismo , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.
Asunto(s)
Hidroliasas/genética , Hidroliasas/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biología Computacional/métodos , Citosol/metabolismo , Hidroximetilglutaril-CoA Sintasa/metabolismo , Mitocondrias/metabolismo , Peroxisomas/metabolismo , Plantas/genética , Plantas/metabolismo , Isoformas de Proteínas/genética , Homología de Secuencia de AminoácidoRESUMEN
Hydroxamate analogs of fosfoxacin, the phosphate homolog of fosmidomycin, have been synthesized and their activity tested on Escherichia coli and Mycobacterium smegmatis DXRs. Except for compound 4b, the IC50 values of phosphate derivatives are approximately 10-fold higher than those of the corresponding phosphonates. Although their inhibitory activity on Escherichia coli DXR is less efficient than their phosphonate analogs, we report the ability of phosphate compounds to inhibit the growth of Escherichia coli. This work points out that the uptake of fosfoxacin and its analogs is taking place via the GlpT and UhpT transporters. As expected, these compounds are inefficient to inhibit the growth of M. smegmatis growth inhibition probably due to a lack of uptake.
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Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Fosfomicina/análogos & derivados , Mycobacterium smegmatis/enzimología , Fosfatos/farmacología , Isomerasas Aldosa-Cetosa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Fosfomicina/síntesis química , Fosfomicina/química , Fosfomicina/farmacología , Estructura Molecular , Fosfatos/química , Relación Estructura-ActividadRESUMEN
S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.
Asunto(s)
Celulasa/metabolismo , Monoterpenos/farmacología , Nicotiana/metabolismo , Prenilación de Proteína/efectos de los fármacos , Sesquiterpenos/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Biomasa , Muerte Celular/efectos de los fármacos , Monoterpenos Ciclohexánicos , Dimetilaliltranstransferasa/antagonistas & inhibidores , Dimetilaliltranstransferasa/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacología , Ácido Mevalónico/farmacología , Modelos Biológicos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Metabolismo Secundario/efectos de los fármacos , Sesquiterpenos/química , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Fosfatos de Azúcar/metabolismo , Nicotiana/citología , Nicotiana/efectos de los fármacos , Nicotiana/enzimologíaRESUMEN
KEY MESSAGE: HMGS functions in phytosterol biosynthesis, development and stress responses. F-244 could specifically-inhibit HMGS in tobacco BY-2 cells and Brassica seedlings. An update on HMGS from higher plants is presented. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is the second enzyme in the mevalonate pathway of isoprenoid biosynthesis and catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce S-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Besides HMG-CoA reductase (HMGR), HMGS is another key enzyme in the regulation of cholesterol and ketone bodies in mammals. In plants, it plays an important role in phytosterol biosynthesis. Here, we summarize the past investigations on eukaryotic HMGS with particular focus on plant HMGS, its enzymatic properties, gene expression, protein structure, and its current status of research in China. An update of the findings on HMGS from animals (human, rat, avian) to plants (Brassica juncea, Hevea brasiliensis, Arabidopsis thaliana) will be discussed. Current studies on HMGS have been vastly promoted by developments in biochemistry and molecular biology. Nonetheless, several limitations have been encountered, thus some novel advances in HMGS-related research that have recently emerged will be touched on.
Asunto(s)
Evolución Molecular , Hidroximetilglutaril-CoA Sintasa/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Ácido Mevalónico/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Brassica/enzimología , China , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Hevea/enzimología , Humanos , Hidroximetilglutaril-CoA Sintasa/química , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Filogenia , Fitosteroles/biosíntesis , Proteínas de Plantas/genética , Ratas , Investigación/tendencias , Homología de Secuencia de AminoácidoRESUMEN
In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.
RESUMEN
Vismione H (VH) is a fluorescent prenylated anthranoid produced by plants from the Hypericaceae family, with antiprotozoal activities against malaria and leishmaniosis. Little is known about its biosynthesis and metabolism in plants or its mode of action against parasites. When VH is isolated from Psorospermum glaberrimum, it is rapidly converted into madagascine anthrone and anthraquinone, which are characterized by markedly different fluorescent properties. To locate the fluorescence of VH in living plant cells and discriminate it from that of the other metabolites, an original strategy combining spectral imaging (SImaging), confocal microscopy, and non-targeted metabolomics using mass spectrometry, was developed. Besides VH, structurally related molecules including madagascine (Mad), emodin (Emo), quinizarin (Qui), as well as lapachol (Lap) and fraxetin (Fra) were analyzed. This strategy readily allowed a spatiotemporal characterization and discrimination of spectral fingerprints from anthranoid-derived metabolites and related complexes with cations and proteins. In addition, our study validates the ability of plant cells to metabolize VH into madagascine anthrone, anthraquinones and unexpected metabolites. These results pave the way for new hypotheses on anthranoid metabolism in plants.
RESUMEN
3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is unique in the first part of the cytoplasmic isoprenoid pathway, as it contains a membrane domain that includes ER-specific retention motifs. When fused to GFP, this domain targets two tobacco BY-2 HMGR isoforms differentially. While the first isoform is ER-localized, a second stress-induced one forms globular structures connected by tubular structures. A serine positioned upstream of the ER retention motif seems to be implicated in this specific subcellular localization. Surprisingly, these structures are closely connected to F-actin, and their intactness is dependent upon the integrity of the filaments or the action of a calmodulin antagonist.
Asunto(s)
Actinas/metabolismo , Hidroximetilglutaril-CoA Reductasas/metabolismo , Nicotiana/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN de Plantas/genética , Retículo Endoplásmico/enzimología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Fracciones Subcelulares/enzimología , Nicotiana/genéticaRESUMEN
Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.
Asunto(s)
Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Antibacterianos/farmacología , Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Fosfomicina/análogos & derivados , Ácidos Hidroxámicos/farmacología , Complejos Multienzimáticos/antagonistas & inhibidores , Organofosfonatos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Terpenos/metabolismo , Antibacterianos/síntesis química , Antimaláricos/síntesis química , Butiratos/síntesis química , Butiratos/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Inhibidores Enzimáticos/síntesis química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Fosfomicina/farmacología , Ácidos Hidroxámicos/síntesis química , Pruebas de Sensibilidad Microbiana , NADP/metabolismo , Organofosfonatos/síntesis químicaRESUMEN
The cytosol-localised mevalonic acid (MVA) pathway delivers the basic isoprene unit isopentenyl diphosphate (IPP). In higher plants, this central metabolic intermediate is also synthesised by the plastid-localised methylerythritol phosphate (MEP) pathway. Both MVA and MEP pathways conspire through exchange of intermediates and regulatory interactions. Products downstream of IPP such as phytosterols, carotenoids, vitamin E, artemisinin, tanshinone and paclitaxel demonstrate antioxidant, cholesterol-reducing, anti-ageing, anticancer, antimalarial, anti-inflammatory and antibacterial activities. Other isoprenoid precursors including isoprene, isoprenol, geraniol, farnesene and farnesol are economically valuable. An update on the MVA pathway and its interaction with the MEP pathway is presented, including the improvement in the production of phytosterols and other isoprenoid derivatives. Such attempts are for instance based on the bioengineering of microbes such as Escherichia coli and Saccharomyces cerevisiae, as well as plants. The function of relevant genes in the MVA pathway that can be utilised in metabolic engineering is reviewed and future perspectives are presented.
Asunto(s)
Butadienos , Hemiterpenos , Ingeniería Metabólica , Ácido Mevalónico , Pentanos , Butadienos/análisis , Butadienos/química , Butadienos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Hemiterpenos/análisis , Hemiterpenos/química , Hemiterpenos/metabolismo , Redes y Vías Metabólicas , Ácido Mevalónico/análisis , Ácido Mevalónico/química , Ácido Mevalónico/metabolismo , Compuestos Organofosforados/análisis , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Pentanos/análisis , Pentanos/química , Pentanos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Little is known about how plant cells regulate the exchange of prenyl diphosphates between the two compartmentalized isoprenoid biosynthesis pathways. Prenylation of proteins is a suitable model to study such interactions between the plastidial methylerythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathways because prenyl moieties used to modify proteins rely on both origins. Tobacco cells expressing a prenylatable GFP were treated with specific MEP and/or MVA pathways inhibitors to block the formation of prenyl diphosphates and therefore the possibility to modify the proteins. Chemical complementation assays using prenyl alcohol precursors restore the prenylation. Indeed, geranylgeraniol (C20 prenyl alcohol) and to a lesser but significant level C15-farnesol restored the prenylation of a protein bearing a geranylgeranylation CaaX motif, which under standard conditions is modified by a MEP-derived prenyl group. However, the restoration takes place in different ways. While geranylgeraniol operates directly as a metabolic precursor, the C15-prenyl alcohol functions indirectly as a signal that leads to shift the metabolic origin of prenyl groups in modified proteins, here from the plastidial MEP pathway in favor of the cytosolic MVA pathway. Furthermore, farnesol interferes negatively with the MEP pathway in an engineered Escherichia coli strain synthesizing isoprenoids either starting from MVA or from MEP. Following the cellular uptake of a fluorescent analog of farnesol, we showed its close interaction with tobacco plastids and modification of plastid homeostasis. As a consequence, in tobacco farnesol supposedly inhibits the plastidial MEP pathway and activates the cytosolic MVA pathway, leading to the shift in the metabolic origin and thereby acts as a potential regulator of crosstalk between the two pathways. Together, those results suggest a new role for farnesol (or a metabolite thereof) as a central molecule for the regulation of isoprenoid biosynthesis in plants.
Asunto(s)
Farnesol/metabolismo , Proteínas de Plantas/metabolismo , Prenilación de Proteína , Línea Celular , Eritritol/análogos & derivados , Eritritol/metabolismo , Proteínas de Plantas/química , Plastidios/metabolismo , Fosfatos de Azúcar/metabolismoRESUMEN
We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.
RESUMEN
A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/análisis , Radiometría/métodos , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Cromatografía en Capa Delgada/métodos , Escherichia coli/genética , Cinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation; and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytosolic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.
Asunto(s)
Línea Celular , Nicotiana/citología , Nicotiana/metabolismo , Esteroles/biosíntesis , Esteroles/metabolismo , Terpenos/metabolismo , Modelos Biológicos , Esteroles/químicaRESUMEN
The mevalonate-independent isoprenoid biosynthesis pathway has been recognized as a promising target for designing new antibiotics. But pathogens treated with compounds such as fosmidomycin, a slow binding inhibitor of 1-deoxy-D-xylulose 5-phosphate reducto-isomerase, the second enzyme in this pathway, develop rapid drug resistance. In Escherichia coli, acquired resistance results mostly from inactivating the cAMP-dependent glpT transporter, thereby preventing import of the inhibitor. Such mutant strains are characterized by cross-resistance to fosfomycin, by susceptibility to efflux pump inhibitors, by disability to use glycerol 3-phosphate as a carbon source or by increased activity of the promoter controlling the expression of the glpABC regulon when grown in presence of fosmidomycin. The quite challenging task consists in conceiving new and efficient inhibitors avoiding resistance acquisition. They should be efficient in blocking the target enzyme, but should also be durably taken up by the organism. To address this issue, it is essential to characterize the mechanisms the pathogen exploits to defeat the antibiotic before resistance is acquired. Having this in mind, a 2-D Fluorescence Difference Gel Electrophoresis proteomic approach has been applied to identify defense responses in E. coli cells being shortly exposed to fosmidomycin (3 h). It seems that combined strategies are promptly induced. The major one consists in preventing toxic effects of the compound either by adapting metabolism and/or by getting rid of the molecule. The strategy adopted by the bacteria is to eliminate the drug from the cell or to increase the tolerance to oxidative stress. The design of new, but still efficient drugs, needs consideration of such rapid modulations required to adapt cell growth in contact of the inhibitor.
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Antibacterianos/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfomicina/análogos & derivados , Pruebas Antimicrobianas de Difusión por Disco , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Fosfomicina/farmacología , Estrés Oxidativo , Fenotipo , Proteoma/metabolismoRESUMEN
Identification of regulatory enzymes is fundamental for engineering metabolic pathways such as the isoprenoid one. All too often, investigation of gene expression remains the major trend in unraveling regulation mechanisms of the isoprenoid cytosolic mevalonate and the plastid-localized methylerythritol phosphate metabolic pathways. But such metabolic regulatory enzymes are frequently multilevel-regulated, especially at a post-translational level. A prominent example is the endoplasmic reticulum-bound 3-hydroxy-3-methylglutaryl coenzyme A reductase catalyzing the synthesis of mevalonic acid. Despite the discovery and the intense efforts made to understand regulation of the methylerythritol phosphate pathway, this enzyme remains a leading player in the regulation of the whole isoprenoid pathway. Strict correlation between this enzyme's gene expression, protein level and enzyme activity is not observed, thus confirming multilevel-regulation. In this context, besides post-translational modifications of proteins, we have to consider feedback of metabolic flow and allosteric regulation, alternative protein structures, targeted proteolysis and/or redox regulation. Such multilevel-regulation processes deliver a range of benefits including rapid response to environmental and physiological challenges or metabolic fluctuations. This review specially emphasizes essential functions of these post-translational events that permit the close regulation of key enzymes involved in plant isoprenoid precursor biosynthesis.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Ácido Mevalónico/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimología , Procesamiento Proteico-Postraduccional , Terpenos/metabolismo , Regulación Alostérica , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Redes y Vías Metabólicas , Oxidación-Reducción , Proteínas de Plantas/genética , Plantas/química , ProteolisisRESUMEN
We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.
RESUMEN
When compared to other organisms, plants are atypical with respect to isoprenoid biosynthesis: they utilize two distinct and separately compartmentalized pathways to build up isoprene units. The co-existence of these pathways in the cytosol and in plastids might permit the synthesis of many vital compounds, being essential for a sessile organism. While substrate exchange across membranes has been shown for a variety of plant species, lack of complementation of strong phenotypes, resulting from inactivation of either the cytosolic pathway (growth and development defects) or the plastidial pathway (pigment bleaching), seems to be surprising at first sight. Hundreds of isoprenoids have been analyzed to determine their biosynthetic origins. It can be concluded that in angiosperms, under standard growth conditions, C20-phytyl moieties, C30-triterpenes and C40-carotenoids are made nearly exclusively within compartmentalized pathways, while mixed origins are widespread for other types of isoprenoid-derived molecules. It seems likely that this coexistence is essential for the interaction of plants with their environment. A major purpose of this review is to summarize such observations, especially within an ecological and functional context and with some emphasis on regulation. This latter aspect still requires more work and present conclusions are preliminary, although some general features seem to exist.
Asunto(s)
Plantas/metabolismo , Terpenos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Redes y Vías Metabólicas , Ácido Mevalónico/química , Ácido Mevalónico/metabolismo , Pentosafosfatos/química , Pentosafosfatos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Feeding tobacco BY-2 cells with [2-(13)C,4-(2)H]deoxyxylulose revealed from the (13)C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% (2)H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. (2)H retention characterizes the isoprene units derived from the DMAPP branch, whereas (2)H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase, the IPP isomerase and the trans-prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with (2)H labeled deoxyxylulose.