RESUMEN
Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high-throughput proteomic studies, mainly focused on post-translational modifications. However, follow-up 'classical' biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein-metabolite interactions, which we consider will become crucial for discoveries in the future.
Asunto(s)
Arabidopsis , Carbono , Carbono/metabolismo , Arabidopsis/metabolismo , Proteómica , Fotosíntesis , Vía de Pentosa Fosfato , Procesamiento Proteico-PostraduccionalRESUMEN
Sugar-alcohols are major photosynthates in plants from the Rosaceae family. Expression of the gene encoding aldose-6-phosphate reductase (Ald6PRase), the critical enzyme for glucitol synthesis in rosaceous species, is regulated by physiological and environmental cues. Additionally, Ald6PRase is inhibited by small molecules (hexose-phosphates and inorganic orthophosphate) and oxidizing compounds. This work demonstrates that Ald6PRase from peach leaves is phosphorylated in planta at the N-terminus. We also show in vitro phosphorylation of recombinant Ald6PRase by a partially purified kinase extract from peach leaves containing Ca2+-dependent protein kinases (CDPKs). Moreover, phosphorylation of recombinant Ald6PRase was inhibited by hexose-phosphates, phosphoenolpyruvate and pyrophosphate. We further show that phosphorylation of recombinant Ald6PRase was maximal using recombinant CDPKs. Overall, our results suggest that phosphorylation could fine-tune the activity of Ald6PRase.
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Prunus persica , Fosforilación , Prunus persica/metabolismo , Fosfatos/metabolismo , Hojas de la Planta/metabolismo , Hexosas/metabolismoRESUMEN
Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long-term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N-acetyl-D-glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP-GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.
Asunto(s)
Agua Potable , Insulinas , Acetilglucosamina/metabolismo , Animales , Femenino , Glucosamina , Glucosa/metabolismo , Glicosilación , Hexosaminas/metabolismo , Insulinas/metabolismo , Longevidad , Masculino , Mamíferos , Ratones , Uridina Difosfato/metabolismoRESUMEN
Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.
Asunto(s)
Fosfatos , Alcoholes del Azúcar , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa/metabolismo , Secuencia de Aminoácidos , Humanos , Manosafosfatos , NADP/metabolismo , Plantas/metabolismo , AzúcaresRESUMEN
Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.
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Glutarredoxinas , Leptospira interrogans , Glutarredoxinas/química , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutatión/metabolismo , Leptospira interrogans/genética , Leptospira interrogans/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Oxidación-Reducción , Compuestos de Sulfhidrilo/química , Tiorredoxinas/metabolismo , Tolueno/análogos & derivadosRESUMEN
Longevity is often associated with stress resistance, but whether they are causally linked is incompletely understood. Here we investigate chemosensory-defective Caenorhabditis elegans mutants that are long-lived and stress resistant. We find that mutants in the intraflagellar transport protein gene osm-3 were significantly protected from tunicamycin-induced ER stress. While osm-3 lifespan extension is dependent on the key longevity factor DAF-16/FOXO, tunicamycin resistance was not. osm-3 mutants are protected from bacterial pathogens, which is pmk-1 p38 MAP kinase dependent, while TM resistance was pmk-1 independent. Expression of P-glycoprotein (PGP) xenobiotic detoxification genes was elevated in osm-3 mutants and their knockdown or inhibition with verapamil suppressed tunicamycin resistance. The nuclear hormone receptor nhr-8 was necessary to regulate a subset of PGPs. We thus identify a cell-nonautonomous regulation of xenobiotic detoxification and show that separate pathways are engaged to mediate longevity, pathogen resistance, and xenobiotic detoxification in osm-3 mutants.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Resistencia a Medicamentos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Longevidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Tunicamicina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Receptores Citoplasmáticos y Nucleares/genética , Factores de Tiempo , Tunicamicina/metabolismoRESUMEN
How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.
Asunto(s)
Arabidopsis/efectos de los fármacos , Dipéptidos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Nicotiana/efectos de los fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulación por Computador , Dipéptidos/química , Dipéptidos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , NADP/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Plantones/efectos de los fármacos , Plantones/metabolismo , Nicotiana/metabolismoRESUMEN
Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. The Integrated Stress Response (ISR) is activated by diverse stimuli, leading to phosphorylation of the eukaryotic translation initiation factor elF2 in its α-subunit. Under replete conditions, elF2 orchestrates 5' cap-dependent mRNA translation and is thus responsible for general protein synthesis. elF2α phosphorylation, the key event of the ISR, reduces global mRNA translation while enhancing the expression of a signature set of stress response genes. Despite the critical role of protein quality control in healthy aging and in numerous longevity pathways, the role of the ISR in longevity remains largely unexplored. ISR activity increases with age, suggesting a potential link with the aging process. Although decreased protein biosynthesis, which occurs during ISR activation, have been linked to lifespan extension, recent data show that lifespan is limited by the ISR as its inhibition extends survival in nematodes and enhances cognitive function in aged mice. Here we survey how aging affects the ISR, the role of the ISR in modulating aging, and pharmacological interventions to tune the ISR. Finally, we will explore the ISR as a plausible target for clinical interventions in aging and age-related disease.
Asunto(s)
Proteínas , Estrés Fisiológico , Animales , Ratones , Estrés Fisiológico/genética , Fosforilación , Transducción de Señal , Envejecimiento/genéticaRESUMEN
Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fosfoenolpiruvato Carboxiquinasa (ATP) , Regulación Alostérica , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Gluconeogénesis , Fosfoenolpiruvato , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , ProteolisisRESUMEN
BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.
Asunto(s)
Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Reductasas/genética , Metionina/metabolismo , Secuencia de Aminoácidos/genética , Catálisis , Citoplasma/enzimología , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimología , Metionina/química , Metionina/genética , Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/ultraestructura , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por SustratoRESUMEN
Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.
RESUMEN
ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Ácido Cítrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorometría/métodos , Fumaratos/metabolismo , Cinética , Malatos/metabolismo , Manganeso/metabolismo , Ácido Oxaloacético/metabolismo , Fotosíntesis , Unión Proteica , Proteínas Recombinantes/metabolismo , Ácido Succínico/metabolismo , Temperatura de TransiciónRESUMEN
Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.
Asunto(s)
Coenzimas/metabolismo , L-Iditol 2-Deshidrogenasa/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Prunus persica/enzimología , Cinética , L-Iditol 2-Deshidrogenasa/química , L-Iditol 2-Deshidrogenasa/genética , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMEN
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Asunto(s)
Aldehído Reductasa/metabolismo , Hojas de la Planta/enzimología , Proteínas de Plantas/metabolismo , Prunus domestica/enzimología , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Fructosadifosfatos/metabolismo , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Fructosafosfatos/farmacología , Glucofosfatos/metabolismo , Glucofosfatos/farmacología , Disulfuro de Glutatión/metabolismo , Hexosafosfatos/metabolismo , Hexosafosfatos/farmacología , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Fosfatos/metabolismo , Fosfatos/farmacología , Filogenia , Hojas de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismoRESUMEN
KEY MESSAGE: The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower. Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.
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Germinación , Helianthus/crecimiento & desarrollo , Helianthus/metabolismo , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Factores de Transcripción/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Metabolismo de los Hidratos de Carbono/genética , Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Germinación/genética , Giberelinas/metabolismo , Gluconeogénesis/genética , Helianthus/genética , Metabolismo de los Lípidos/genética , Modelos Biológicos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Plantones/metabolismo , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H2O2 (Chyp 1% ~30 µM at pH 7.0 and 30°C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.
Asunto(s)
Proteínas Bacterianas/química , Leptospira interrogans/enzimología , Peroxirredoxinas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxirredoxinas/biosíntesis , Peroxirredoxinas/genética , FilogeniaRESUMEN
Glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. Herein we report the molecular cloning, heterologous expression and characterization of Gol dehydrogenase (GolDHase, EC 1.1.1.14) from peach (Prunus persica) fruits. The recombinant enzyme showed kinetic parameters similar to those reported for orthologous enzymes purified from apple and pear fruits. The activity of recombinant GolDHase was strongly inhibited by Cu(2+) and Hg(2+), suggesting that it might have cysteine residues critical for functionality. Oxidizing compounds (such as diamide, hydrogen peroxide and oxidized glutathione) inactivated the enzyme, whereas its activity was restored after incubation with reduced glutathione and thioredoxin from Escherichia coli. Recombinant thioredoxin h from peach fruits also recovered the activity of oxidized GolDHase. Our results suggest that peach fruit GolDHase could be redox regulated in vivo and this would be of relevance to determine carbon assimilation and partitioning in plants accumulating sugar alcohols.