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1.
Biochem J ; 477(6): 1149-1158, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32150261

RESUMO

Searching for compounds that inhibit the growth of photosynthetic organisms highlighted a prominent effect at micromolar concentrations of the nitroheteroaromatic thioether, 2-nitrothiophene, applied in the light. Since similar effects were reminiscent to those obtained also by radicals produced under excessive illumination or by herbicides, and in light of its redox potential, we suspected that 2-nitrothiophene was reduced by ferredoxin, a major reducing compound in the light. In silico examination using docking and tunneling computing algorithms of the putative interaction between 2-nitrothiophene and cyanobacterial ferredoxin has suggested a site of interaction enabling robust electron transfer from the iron-sulfur cluster of ferredoxin to the nitro group of 2-nitrothiophene. ESR and oximetry analyses of cyanobacterial cells (Anabaena PCC7120) treated with 50 µM 2-nitrothiophene under illumination revealed accumulation of oxygen radicals and peroxides. Gas chromatography mass spectrometry analysis of 2-nitrothiophene-treated cells identified cytotoxic nitroso and non-toxic amino derivatives. These products of the degradation pathway of 2-nitrohiophene, which initializes with a single electron transfer that forms a short-live anion radical, are then decomposed to nitrate and thiophene, and may be further reduced to a nitroso hydroxylamine and amino derivatives. This mechanism of toxicity is similar to that of nitroimidazoles (e.g. ornidazole and metronidazole) reduced by ferredoxin in anaerobic bacteria and protozoa, but differs from that of ornidazole in planta.

3.
Biochem J ; 473(23): 4413-4426, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27647935

RESUMO

Ornidazole of the 5-nitroimidazole drug family is used to treat protozoan and anaerobic bacterial infections via a mechanism that involves preactivation by reduction of the nitro group, and production of toxic derivatives and radicals. Metronidazole, another drug family member, has been suggested to affect photosynthesis by draining electrons from the electron carrier ferredoxin, thus inhibiting NADP+ reduction and stimulating radical and peroxide production. Here we show, however, that ornidazole inhibits photosynthesis via a different mechanism. While having a minute effect on the photosynthetic electron transport and oxygen photoreduction, ornidazole hinders the activity of two Calvin cycle enzymes, triose-phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Modeling of ornidazole's interaction with ferredoxin of the protozoan Trichomonas suggests efficient electron tunneling from the iron-sulfur cluster to the nitro group of the drug. A similar docking site of ornidazole at the plant-type ferredoxin does not exist, and the best simulated alternative does not support such efficient tunneling. Notably, TPI was inhibited by ornidazole in the dark or when electron transport was blocked by dichloromethyl diphenylurea, indicating that this inhibition was unrelated to the electron transport machinery. Although TPI and GAPDH isoenzymes are involved in glycolysis and gluconeogenesis, ornidazole's effect on respiration of photoautotrophs is moderate, thus raising its value as an efficient inhibitor of photosynthesis. The scarcity of Calvin cycle inhibitors capable of penetrating cell membranes emphasizes on the value of ornidazole for studying the regulation of this cycle.


Assuntos
Bactérias Anaeróbias/efeitos dos fármacos , Ornidazol/farmacologia , Fotossíntese/efeitos dos fármacos , Cianobactérias/efeitos dos fármacos , Ferredoxinas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Metronidazol/farmacologia , Modelos Biológicos , Synechocystis/efeitos dos fármacos , Trichomonas/efeitos dos fármacos , Trichomonas/metabolismo , Triose-Fosfato Isomerase/metabolismo
4.
New Phytol ; 197(1): 177-85, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23078356

RESUMO

Diatoms are important players in the global carbon cycle. Their apparent photosynthetic affinity for ambient CO(2) is much higher than that of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), indicating that a CO(2)-concentrating mechanism (CCM) is functioning. However, the nature of the CCM, a biophysical or a biochemical C(4), remains elusive. Although (14)C labeling experiments and presence of complete sets of genes for C(4) metabolism in two diatoms supported the presence of C(4), other data and predicted localization of the decarboxylating enzymes, away from Rubisco, makes this unlikely. We used RNA-interference to silence the single gene encoding pyruvate-orthophosphate dikinase (PPDK) in Phaeodactylum tricornutum, essential for C(4) metabolism, and examined the photosynthetic characteristics. The mutants possess much lower ppdk transcript and PPDK activity but the photosynthetic K(1/2) (CO(2)) was hardly affected, thus clearly indicating that the C(4) route does not serve the purpose of raising the CO(2) concentration in close proximity of Rubisco in P. tricornutum. The photosynthetic V(max) was slightly reduced in the mutant, possibly reflecting a metabolic constraint that also resulted in a larger lipid accumulation. We propose that the C(4) metabolism does not function in net CO(2) fixation but helps the cells to dissipate excess light energy and in pH homeostasis.


Assuntos
Dióxido de Carbono/metabolismo , Carbono/metabolismo , Diatomáceas/enzimologia , Fotossíntese , Piruvato Ortofosfato Diquinase/genética , Ciclo do Carbono , Radioisótopos de Carbono/metabolismo , Clonagem Molecular , Diatomáceas/genética , Diatomáceas/crescimento & desenvolvimento , Ativação Enzimática , Ensaios Enzimáticos , Fluorescência , Concentração de Íons de Hidrogênio , Luz , Metabolismo dos Lipídeos , Plasmídeos/genética , Plasmídeos/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Interferência de RNA , Espectroscopia de Infravermelho com Transformada de Fourier , Transcrição Genética
5.
J Exp Bot ; 62(12): 4173-82, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21551078

RESUMO

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.


Assuntos
Mutagênese , Fotossíntese/genética , Ribulose-Bifosfato Carboxilase/genética , Synechocystis/enzimologia , Synechocystis/crescimento & desenvolvimento , Substituição de Aminoácidos/genética , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Clorofila/metabolismo , Ativação Enzimática/efeitos dos fármacos , Cinética , Mutagênese/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Oxigênio/metabolismo , Fosfatos/farmacologia , Fotossíntese/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ribulose-Bifosfato Carboxilase/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/genética , Synechocystis/ultraestrutura
6.
Photosynth Res ; 90(3): 233-42, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17286188

RESUMO

Nuphar lutea is an amphibious plant with submerged and aerial foliage, which raises the question how do both leaf types perform photosynthetically in two different environments. We found that the aerial leaves function like terrestrial sun-leaves in that their photosynthetic capability was high and saturated under high irradiance (ca. 1,500 mumol photons m(-2) s(-1)). We show that stomatal opening and Rubisco activity in these leaves co-limited photosynthesis at saturating irradiance fluctuating in a daily rhythm. In the morning, sunlight stimulated stomatal opening, Rubisco synthesis, and the neutralization of a night-accumulated Rubisco inhibitor. Consequently, the light-saturated quantum efficiency and rate of photosynthesis increased 10-fold by midday. During the afternoon, gradual closure of the stomata and a decrease in Rubisco content reduced the light-saturated photosynthetic rate. However, at limited irradiance, stomatal behavior and Rubisco content had only a marginal effect on the photosynthetic rate, which did not change during the day. In contrast to the aerial leaves, the photosynthesis rate of the submerged leaves, adapted to a shaded environment, was saturated under lower irradiance. The light-saturated quantum efficiency of these leaves was much lower and did not change during the day. Due to their low photosynthetic affinity for CO(2) (35 muM) and inability to utilize other inorganic carbon species, their photosynthetic rate at air-equilibrated water was CO(2)-limited. These results reveal differences in the photosynthetic performance of the two types of Nuphar leaves and unravel how photosynthetic daily rhythm in the aerial leaves is controlled.


Assuntos
Ritmo Circadiano/fisiologia , Nuphar/fisiologia , Fotossíntese/fisiologia , Folhas de Planta/fisiologia , Ribulose-Bifosfato Carboxilase/fisiologia , Luz , Áreas Alagadas
7.
J Bacteriol ; 187(12): 4222-8, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15937184

RESUMO

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.


Assuntos
Fosfatos/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo , Synechocystis/enzimologia , Sítios de Ligação , Catálise , Ativação Enzimática , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fotossíntese/genética , Fotossíntese/fisiologia , Conformação Proteica , Synechocystis/genética
8.
J Bacteriol ; 185(5): 1509-17, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12591867

RESUMO

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Cisteína/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Alquilantes/química , Proteínas de Bactérias/genética , Cianobactérias/genética , Cisteína/genética , Estabilidade Enzimática , Iodoacetatos/química , Cinética , Nitrogênio/metabolismo , Oxirredução , Pentosefosfatos/metabolismo , Mutação Puntual , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/genética
9.
Plant Biotechnol J ; 1(1): 43-50, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17147679

RESUMO

Transgenic Arabidopsis thaliana and Nicotiana tabacum plants that express ictB, a gene involved in HCO3- accumulation within the cyanobacterium Synechococcus sp. PCC 7942, exhibited significantly faster photosynthetic rates than the wild-types under limiting but not under saturating CO2 concentrations. Under conditions of low relative humidity, growth of the transgenic A. thaliana plants was considerably faster than the wild-type. This enhancement of growth was not observed under humid conditions. There was no difference in the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) detected in the wild-types and their respective transgenic plants. Following activation in vitro, the activities of RubisCO from either low- or high-humidity-grown transgenic plants were similar to those observed in the wild-types. In contrast, the in vivo RubisCO activity, i.e. without prior activation, in plants grown under low humidity was considerably higher in ictB-expressing plants than in their wild-types. The CO2 compensation point in the transgenic plants that express ictB was lower than in the wild-types, suggesting that the concentration of CO2 in close proximity to RubisCO was higher. This may explain the higher activation level of RubisCO and enhanced photosynthetic activities and growth in the transgenic plants. These data indicated a potential use of ictB for the stimulation of crop yield.

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