RESUMEN
Rubisco catalyzes the fixation of CO2 into organic compounds that are used for plant growth and the production of agricultural products, and specific sugar-phosphate derivatives bind tightly to the active sites of Rubisco, locking the enzyme in a catalytically inactive conformation. 2-carboxy-d-arabinitol-1-phosphate phosphatase (CA1Pase) dephosphorylates such tight-binding inhibitors, contributing to the maintenance of Rubisco activity. Here, we investigated the hypothesis that overexpressing ca1pase would decrease the abundance of Rubisco inhibitors, thereby increasing the activity of Rubisco and enhancing photosynthetic performance and productivity in wheat (Triticum aestivum). Plants of four independent wheat transgenic lines overexpressing ca1pase showed up to 30-fold increases in ca1pase expression compared to the wild type. Plants overexpressing ca1pase had lower numbers of Rubisco tight-binding inhibitors and higher Rubisco activation state than the wild type; however, there were 17% to 60% fewer Rubisco active sites in the four transgenic lines than in the wild type. The lower Rubisco content in plants overexpressing ca1pase resulted in lower initial and total carboxylating activities measured in flag leaves at the end of the vegetative stage and lower aboveground biomass and grain yield measured in fully mature plants. Hence, contrary to what would be expected, ca1pase overexpression decreased Rubisco content and compromised wheat grain yields. These results support a possible role for Rubisco inhibitors in protecting the enzyme and maintaining an adequate number of Rubisco active sites to support carboxylation rates in planta.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Triticum/enzimología , Biomasa , Plantas Modificadas Genéticamente , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Triticum/genética , Triticum/crecimiento & desarrolloRESUMEN
The photosynthetic CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) is inhibited by nonproductive binding of its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. Reactivation requires ATP-hydrolysis-powered remodeling of the inhibited complexes by diverse molecular chaperones known as rubisco activases (Rcas). Eukaryotic phytoplankton of the red plastid lineage contain so-called red-type rubiscos, some of which have been shown to possess superior kinetic properties to green-type rubiscos found in higher plants. These organisms are known to encode multiple homologs of CbbX, the α-proteobacterial red-type activase. Here we show that the gene products of two cbbX genes encoded by the nuclear and plastid genomes of the red algae Cyanidioschyzon merolae are nonfunctional in isolation, but together form a thermostable heterooligomeric Rca that can use both α-proteobacterial and red algal-inhibited rubisco complexes as a substrate. The mechanism of rubisco activation appears conserved between the bacterial and the algal systems and involves threading of the rubisco large subunit C terminus. Whereas binding of the allosteric regulator RuBP induces oligomeric transitions to the bacterial activase, it merely enhances the kinetics of ATP hydrolysis in the algal enzyme. Mutational analysis of nuclear and plastid isoforms demonstrates strong coordination between the subunits and implicates the nuclear-encoded subunit as being functionally dominant. The plastid-encoded subunit may be catalytically inert. Efforts to enhance crop photosynthesis by transplanting red algal rubiscos with enhanced kinetics will need to take into account the requirement for a compatible Rca.
Asunto(s)
Proteínas de Plantas/metabolismo , Rhodophyta/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Regulación Alostérica/fisiología , Cinética , Chaperonas Moleculares/metabolismo , Fotosíntesis/genética , Fotosíntesis/fisiología , Proteínas de Plantas/genética , Plastidios/genética , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosafosfatos/metabolismoRESUMEN
The activity of the photosynthetic carbon-fixing enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), is partially inhibited by arsenite in the millimolar concentration range. However, micromolar arsenite can fully inhibit Rubisco in the presence of a potentiating monothiol such as cysteine, cysteamine, 2-mercaptoethanol or N-acetylcysteine, but not glutathione. Arsenite reacts specifically with the vicinal Cys172-Cys192 from the large subunit of Rubisco and with the monothiol to establish a ternary complex, which is suggested to be a trithioarsenical. The stability of the complex is strongly dependent on the nature of the monothiol. Enzyme activity is fully recovered through the disassembly of the complex after eliminating arsenite and/or the thiol from the medium. The synergic combination of arsenite and a monothiol acts also in vivo stopping carbon dioxide fixation in illuminated cultures of Chlamydomonas reinhardtii. Again, this effect may be reverted by washing the cells. However, in vivo inhibition does not result from the blocking of Rubisco since mutant strains carrying Rubiscos with Cys172 and/or Cys192 substitutions (which are insensitive to arsenite in vitro) are also arrested. This suggests the existence of a specific sensor controlling carbon fixation that is even more sensitive than Rubisco to the arsenite-thiol synergism.
Asunto(s)
Arsenitos/toxicidad , Chlamydomonas reinhardtii/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Compuestos de Sulfhidrilo/farmacología , Chlamydomonas reinhardtii/enzimología , Sinergismo FarmacológicoRESUMEN
This study investigated the effects of cinnamic acid (CA) on ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and the endogenous polyamine levels of cowpea leaves. The results show that 0.1 mM CA treatment decreased photosynthetic rate (P(n)) and RuBPC activity, but it did not affect the maximal photochemical efficiency of PSII (F(v)/F(m)), the actual photochemical efficiency of PSII (PhiPSII), intercellular CO(2) concentration (C(i)), and relative chlorophyll content. These suggest that the decrease in P(n) is at least partially attributed to a lowered RuBPC activity. In addition, 0.1 mM CA treatment increased the putrescine (Put) level, but decreased spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put (PAs) ratio in the leaves. The exogenous application of 1 mM Spd markedly reversed these CA-induced effects for polyamine and partially restored the PAs ratio and RuBPC activity in leaves. Methylglyoxal-bis (guanylhydrazone) (MGBG), which is an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), results in the inability of activated cells to synthesize Spd and exacerbates the negative effects induced by CA. The exogenous application of 1 mM D-arginine (D-Arg), which is an inhibitor of Put biosynthesis, decreased the levels of Put, but increased the PAs ratio and RuBPC activity in leaves. These results suggest that 0.1 mM CA inhibits RuBPC activity by decreasing the levels of endogenous free and perchloric acid soluble (PS) conjugated Spm, as well as the PAs ratio.
Asunto(s)
Cinamatos/farmacología , Fabaceae/efectos de los fármacos , Fabaceae/enzimología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Espermina/metabolismo , Arginina/farmacología , Modelos Lineales , Malondialdehído/metabolismo , Mitoguazona/farmacología , Percloratos/metabolismo , Fotosíntesis/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/fisiología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Solubilidad/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
The aim of this study was to quantify the effect of the pollutant, trifluoroacetate (TFA), on growth and photosynthesis of Phaseolus vulgaris (C(3)) and Zea mays (C(4)) in order to elucidate the physiological and biochemical basis of its inhibitory action. In whole plant studies, photosynthetic gas exchange, fast phase fluorescence kinetics and Rubisco activity were measured in parallel over a 14-day period in plants cultivated in a water culture system with NaTFA added at concentrations ranging from 0.625 to 160mgl(-1). Although initial stimulation of some photosynthetic parameters was observed at low TFA concentrations early on in the experiment, marked inhibition occurred at higher concentrations. In general Z. mays was affected more severely than P. vulgaris showing a large TFA-induced decrease in both apparent carboxylation efficiency (ACE) and in vitro Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) activity. Analysis of photosynthetic gas exchange revealed that besides constraints on mesophyll processes such as Rubisco activity, stomatal limitation also increased with increasing TFA concentration, especially in P. vulgaris. In depth analysis of the fast phase fluorescence transients pointed at TFA-induced uncoupling of the oxygen evolving complex (OEC) and inhibition of electron transport beyond Q(a) including possible constraints on the reduction of end electron acceptors of photosystem I.
Asunto(s)
Dióxido de Carbono/metabolismo , Phaseolus/efectos de los fármacos , Fotosíntesis/efectos de los fármacos , Estomas de Plantas/fisiología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ácido Trifluoroacético/toxicidad , Zea mays/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Hidrocarburos Fluorados/metabolismo , Oxígeno/metabolismo , Phaseolus/crecimiento & desarrollo , Phaseolus/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
Nitric oxide (NO) is a signaling molecule that affects a myriad of processes in plants. However, the mechanistic details are limited. NO post-translationally modifies proteins by S-nitrosylation of cysteines. The soluble S-nitrosoproteome of a medicinal, crassulacean acid metabolism (CAM) plant, Kalanchoe pinnata, was purified using the biotin switch technique. Nineteen targets were identified by MALDI-TOF mass spectrometry, including proteins associated with carbon, nitrogen and sulfur metabolism, the cytoskeleton, stress and photosynthesis. Some were similar to those previously identified in Arabidopsis thaliana, but kinesin-like protein, glycolate oxidase, putative UDP glucose 4-epimerase and putative DNA topoisomerase II had not been identified as targets previously for any organism. In vitro and in vivo nitrosylation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), one of the targets, was confirmed by immunoblotting. Rubisco plays a central role in photosynthesis, and the effect of S-nitrosylation on its enzymatic activity was determined using NaH14CO3. The NO-releasing compound S-nitrosoglutathione inhibited its activity in a dose-dependent manner suggesting Rubisco inactivation by nitrosylation for the first time.
Asunto(s)
Kalanchoe/metabolismo , Extractos Vegetales/química , Plantas Medicinales/química , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/química , Arabidopsis/metabolismo , Bioquímica/métodos , Carbono/química , Cisteína/química , ADN-Topoisomerasas de Tipo II/química , Inhibidores Enzimáticos/farmacología , Nitrógeno/química , Fotosíntesis , Proteínas de Plantas/química , Ribulosa-Bifosfato Carboxilasa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , UDPglucosa 4-Epimerasa/químicaRESUMEN
In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/metabolismo , Productos Agrícolas/enzimología , Inducción Enzimática , Unión Proteica , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) of a thermophilic cyanobacterium, Thermosynechococcus vulcanus, was cloned and expressed in Escherichia coli. The purified enzyme had higher thermostability than RuBisCOs isolated from mesophilic cyanobacteria. Prediction of the tertiary structure was performed using the software Molecular Operating Environment (MOE). The predicted structure did not give any clue about the basis of thermostability. Then, the molecular docking of substrates and inhibitors in the catalytic site were carried out to test analogs for consistency of ribulose 1,5-bisphosphate, a RuBisCO substrate. The analogs were searched in the Kyoto Encyclopedia of Genes and Genomes (KEGG), and 99 compounds were selected for the docking. The mol files from LIGAND Database in KEGG were changed to a three dimensional (3D) structure for use in docking simulation. The docking simulation was performed on ASEDock of MOE, and the SiteFinder command suggested about 20 candidates for the docking site of the compounds. Based on the homology of these candidate sites with the xylulose 1,5-bisphosphate (XBP)-binding site of RuBisCO isolated from Synechococcus PCC 6301, one site was selected for the docking simulation. The 40 compounds with the highest docking energies included synthetic organic substances that had never been demonstrated to be inhibitors of RuBisCO. The total docking energies were -102 kcal/mol, -104 kcal/mol, -94.0 kcal/mol, and -57.7 kcal/mol for ribulose 1,5-bisphosphate (RuBP), etidronate, risedronate, and citrate respectively. Kinetic analysis of RuBisCO revealed a K(m) value of 315 microM for RuBP, and K(i) values of 1.70, 0.93, and 2.04 mM for etidronate, risedronate, and citrate respectively. From these values, the binding energies were estimated to be -4.85, -3.84, -4.20, and -3.73 kcal/mol for RuBP, etidronate, risedronate, and citrate respectively. The differences between the values estimated from experimental data and by simulation may mainly depend on the dissimilarity of the environment for the protein and ligands between the experiments and the simulation. The results obtained here suggested a few new inhibitors, which might be useful as tools for studying the relationship between the structure and the function of RuBisCO.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/aislamiento & purificación , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Synechococcus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Clonación Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Ligandos , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Ribulosa-Bifosfato Carboxilasa/biosíntesis , Ribulosa-Bifosfato Carboxilasa/química , Relación Estructura-ActividadRESUMEN
Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure of a small loop (between Ser61 and Thr68) when oxidized. Progressive oxidation of Rubisco cysteines using disulphide/thiol mixtures at different ratios have shown that inactivation occurs under milder oxidative conditions than proteolytic sensitization, suggesting the involvement of different critical cysteines. Site-directed mutagenesis of conserved cysteines in the Chlamydomonas reinhardtii Rubisco identified Cys449 and Cys459 among those involved in oxidative inactivation, and Cys172 and Cys192 as the specific target for arsenite. The physiological importance of Rubisco redox regulation is supported by the in vivo response of the cysteine mutants to stress conditions. Substitution of Cys172 caused a pronounced delay in stress-induced Rubisco degradation, while the replacement of the functionally redundant Cys449-Cys459 pair resulted in an enhanced catabolism with a faster high-molecular weight polymerization and translocation to membranes. These results suggest that several cysteines contribute to a sequence of conformational changes that trigger the different stages of Rubisco catabolism under increasing oxidative conditions.
Asunto(s)
Cisteamina/química , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/metabolismo , Secuencia de Aminoácidos , Cloroplastos/enzimología , Oxidación-Reducción , Células Vegetales , Plantas/enzimología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidoresRESUMEN
During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.
Asunto(s)
Proteínas Bacterianas/metabolismo , Nicotiana/enzimología , Proteínas de Plantas/metabolismo , Rhodophyta/enzimología , Rhodospirillum rubrum/enzimología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosafosfatos/metabolismo , Synechococcus/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Catálisis , Ácidos Glicéricos/metabolismo , Ligandos , Organofosfatos/metabolismo , Oxidación-Reducción , Pentosafosfatos/metabolismo , Pentosafosfatos/farmacología , Filogenia , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/aislamiento & purificación , Unión Proteica , Piruvatos/metabolismo , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/clasificación , Ribulosa-Bifosfato Carboxilasa/aislamiento & purificación , Especificidad de la Especie , Spinacia oleracea/enzimología , Alcoholes del Azúcar/metabolismo , Alcoholes del Azúcar/farmacología , Fosfatos de Azúcar/metabolismoRESUMEN
The effect of temperature, O(2) and Mg(++) on the kinetic characteristics of the slow inactivation (fallover) of Rubisco isolated from spinach (Spinacia oleracea L.) was determined. Comparing 25 and 45 degrees C, the rate of activity decline of Rubisco increased by 20-fold, but the final ratio of steady state to initial activity increased from 0.38 to 0.62, respectively. Low CO(2) increased the extent of fallover but only caused a marginal increase in fallover rate in agreement with results reported previously. In contrast, increased O(2) during catalysis significantly increased only the fallover rate. Low Mg(++) greatly increased the fallover of Rubisco both in rate and extent. Rubisco carbamylation was assayed using a new separation technique and it revealed that a loss of carbamylation largely accounted for the increased fallover observed with low Mg(++). In conclusion, Rubisco fallover is facilitated by high temperature, low concentration of CO(2) or Mg(++), and high O(2). The physiological importance of these factors in affecting Rubisco fallover and contributing to photosynthetic inhibition at high temperatures in planta are discussed.
Asunto(s)
Magnesio/farmacología , Oxígeno/farmacología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/metabolismo , Spinacia oleracea/enzimología , Temperatura , Catálisis , Relación Dosis-Respuesta a Droga , CinéticaRESUMEN
Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.
Asunto(s)
Ácido Ascórbico/farmacología , Endopeptidasas/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismo , Western Blotting , Radioisótopos de Carbono , Quimotripsina/metabolismo , Estabilidad de Enzimas/efectos de los fármacos , Glutatión/farmacología , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Guanidinas/farmacología , Inmunoquímica , Lisina/farmacología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/químicaRESUMEN
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is modulated in vivo either by reaction with CO2 and Mg2+ to carbamylate a lysine residue in the catalytic site, or by the binding of inhibitors within the catalytic site. Binding of inhibitors blocks either activity or the carbamylation of the lysine residue that is essential for activity. At night, in many species, 2-carboxyarabinitol-1-phosphate (CA1P) is formed which binds tightly to Rubisco, inhibiting catalytic activity. Recent work has shown that tight-binding inhibitors can also decrease Rubisco activity in the light and contribute to the regulation of Rubisco activity. Here we determine the influence that such inhibitors of Rubisco exert on catalytic activity during drought stress. In tobacco plants, 'total Rubisco activity', i.e. the activity following pre-incubation with CO2 and Mg2+, was positively correlated with leaf relative water content. However, 'total Rubisco activity' in extracts from leaves with low water potential increased markedly when tightly bound inhibitors were removed, thus increasing the number of catalytic sites available. This suggests that in tobacco the decrease of Rubisco activity under drought stress is not primarily the result of changes in activation by CO2 and Mg2+ but due rather to the presence of tight-binding inhibitors. The amounts of inhibitor present in leaves of droughted tobacco based on the decrease in Rubisco activity per mg soluble protein were usually much greater than the amounts of the known inhibitors (CA1P and 'daytime inhibitor') that can be recovered in acid extracts. Alternative explanations for the difference between maximal and total activities are discussed.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa/metabolismo , Agua/fisiología , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacología , Clorofila/fisiología , Desastres , Magnesio/farmacología , Presión Osmótica , Pentosafosfatos/metabolismo , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/efectos de los fármacos , Estrés Mecánico , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , Triticum/efectos de los fármacos , Triticum/metabolismo , Agua/farmacologíaRESUMEN
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function. In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae. To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes. Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function. Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values. The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity. Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.
Asunto(s)
Sustitución de Aminoácidos/genética , Arginina/genética , Dióxido de Carbono/metabolismo , Cloroplastos/enzimología , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Ribulosa-Bifosfato Carboxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Estabilidad de Enzimas/genética , Calor , Cinética , Datos de Secuencia Molecular , Fenotipo , Estructura Secundaria de Proteína/genética , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Especificidad por Sustrato/genética , Transformación GenéticaRESUMEN
Proline caused irreversible inhibition (involving reduction in V(max) without altering K(m) for RuBP) in Rubisco activity. Proline-induced suppression in Rubisco activity did not exceed beyond approximately 65% of the original activity even upon exposure to higher levels of proline for prolonged duration. However, NaCl-induced reduction in Rubisco activity was reversible. Native PAGE analysis of Rubisco-incubated with proline showed the presence of two distinct bands corresponding to approximately 430 and approximately 28 kDa, but that incubated with NaCl showed a single band. SDS-PAGE analysis revealed that the approximately 430- and approximately 28-kDa bands represent octamers of large subunits and dimers of small subunits, respectively. These results demonstrated for the first time that proline suppresses Rubisco activity by bringing about dissociation of the small subunits from the octamer core of large subunits, probably by weakening hydrophobic interactions between them.
Asunto(s)
Prolina/metabolismo , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Ribulosa-Bifosfato Carboxilasa/química , Ribulosa-Bifosfato Carboxilasa/metabolismo , Rosales/enzimologíaRESUMEN
In order to identify functionally important amino acid residues in the chloroplast protein import machinery, chloroplasts were preincubated with amino-acid-modifying reagents and then allowed to import or form early import intermediates with precursor proteins. Incubation of chloroplasts with N-ethyl maleimide, diethyl pyrocarbonate, phenylglyoxal, 4,4'-di-isothiocyanatostilbene 2,2'-disulphonic acid (DIDS), dicyclohexylcarbodiimide (DCCD), and 1-ethyl- 3-dimethylaminopropylcarbodiimide (EDC) inhibited both import and formation of early import intermediates with precursor proteins by chloroplasts. This suggests that one or more of the binding components of the chloroplast protein import machinery contains functionally important solvent-exposed cysteine, histidine, arginine, and aspartate/glutamate residues, as well as functionally important lysine and aspartate/ glutamate residues in a hydrophobic environment.
Asunto(s)
Cloroplastos/metabolismo , Inhibidores Enzimáticos/farmacología , Ferredoxina-NADP Reductasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Precursores de Proteínas/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Aminoácidos , Proteínas de Cloroplastos , Cloroplastos/efectos de los fármacos , Reactivos de Enlaces Cruzados , Diciclohexilcarbodiimida/antagonistas & inhibidores , Diciclohexilcarbodiimida/farmacocinética , Dietil Pirocarbonato/farmacología , Electroforesis , Etildimetilaminopropil Carbodiimida/análogos & derivados , Etildimetilaminopropil Carbodiimida/farmacología , Etilmaleimida/farmacología , Ferredoxina-NADP Reductasa/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Pisum sativum/metabolismo , Fenilglioxal/farmacología , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/metabolismo , Plásmidos , Precursores de Proteínas/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-a-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 degrees C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 degrees C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO2 and Mg2+. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 degrees C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 degrees C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO2 (Ci). However, the inhibition of the CER by nigericin, a K+ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco.
Asunto(s)
Ribulosa-Bifosfato Carboxilasa/metabolismo , Dióxido de Carbono/metabolismo , Activación Enzimática , Gossypium/enzimología , Calor , Luz , Hojas de la Planta/enzimología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , TemperaturaRESUMEN
Trypsin-catalysed cleavage of purified ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the resultant irreversible loss of carboxylase activity were prevented by prior incubation with the naturally occurring nocturnal Rubisco inhibitor 2'-carboxy-D-arabitinol 1-phosphate (CA1P), as well as with ribulose 1,5-bisphosphate (RuBP), Mg2+ and CO2. CA1P also protected Rubisco from loss of activity caused by carboxypeptidase A. When similar experiments were carried out using soluble chloroplast proteases, CA1P was again able to protect Rubisco against proteolytic degradation and the consequent irreversible loss of catalytic activity. Thus, CA1P prevents the proteolytic breakdown of Rubisco by endogenous and exogenous proteases. In this way, CA1P may affect the amounts of Rubisco protein available for photosynthetic CO2 assimilation. Rubisco turnover (in the presence of RuBP, Mg2+ and CO2) may confer similar protection against proteases in the light.
Asunto(s)
Pentosafosfatos/farmacología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Carboxipeptidasas/metabolismo , Carboxipeptidasas A , Dominio Catalítico , Cloroplastos/enzimología , Endopeptidasas/metabolismo , Inhibidores Enzimáticos/farmacología , Cinética , Pentosafosfatos/metabolismo , Plantas Medicinales , Plantas Tóxicas , Rhamnus/enzimología , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Triticum/enzimología , Tripsina/farmacologíaRESUMEN
A recent controversial report suggests that the nocturnal inhibitor of Rubisco, 2-carboxy-D-arabinitol 1-phosphate (CAIP), does not bind to Rubisco in vivo and therefore that CA1P has no physiological relevance to photosynthetic regulation. It is now proved that a direct rapid assay can be used to distinguish between Rubisco-bound and free CA1P, as postulated in the controversial report. Application of this direct assay demonstrates that CA1P is bound to Rubisco in vivo in dark-adapted leaves. Furthermore, CA1P is shown to be in the chloroplasts of mesophyll cells. Thus, CA1P does play a physiological role in the regulation of Rubisco.
Asunto(s)
Fabaceae/enzimología , Pentosafosfatos/metabolismo , Hojas de la Planta/metabolismo , Plantas Medicinales , Ribulosa-Bifosfato Carboxilasa/antagonistas & inhibidores , Sitios de Unión , Clorofila/análisis , Cloroplastos/química , Cloroplastos/enzimología , Cloroplastos/metabolismo , Inhibidores Enzimáticos/metabolismo , Fabaceae/metabolismo , Liofilización , Luz , Fotoperiodo , Hojas de la Planta/enzimología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Sulfatos/farmacología , Factores de TiempoRESUMEN
Seedlings of Brassica juncea, Sesbania sesban, and Oryza sativa exposed to salt stress accumulated proline to levels as high as 4- to 20-fold over those of controls. Because chloroplasts are the major site for synthesis of stress induced proline accumulation, in vitro studies were conducted to see how the over-accumulation of this solute influences the activity of the major chloroplastic enzyme Ribulose 1,5-bis-phosphate carboxylase (Rubisco) purified from the above plant species. Surprisingly, proline (believed to be a compatible solute) suppressed the activity of Rubisco significantly even when present at a concentration as low as 100 mM. The extent of this inhibition in Rubisco activity increased with an increase in the concentration of proline. Irrespective of the plant species from which Rubisco was purified, its activity declined by about 50% in the presence of 1 M proline. Rubisco from all three plant species was sensitive to NaCl and proline accelerated salt induced suppression in its activity. To the best of our knowledge this is the first report wherein a negative effect of proline (which is believed to protect enzymes under stress) has been clearly demonstrated. This perturbing effect of proline on Rubisco from higher plant species belonging to three distinct families cautions targeting of gene(s)/gene products for over-production of proline into chloroplasts.