RESUMO
Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.
Assuntos
Cisteína/metabolismo , Diatomáceas/enzimologia , Fosfoglicerato Quinase/metabolismo , Plastídeos/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Diamida/farmacologia , Diatomáceas/genética , Diatomáceas/fisiologia , Dissulfetos/metabolismo , Ditiotreitol/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Glutationa/farmacologia , Peróxido de Hidrogênio/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nitroprussiato/farmacologia , Oxirredução , Fosfoglicerato Quinase/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Plastídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Ácidos Sulfênicos/metabolismo , Tiorredoxinas/farmacologiaRESUMO
The aim of this study was to assess the combined effects of temperature and UVR on the photosynthesis performance of two diatoms -Chaetoceros gracilis and Thalassiosira weissflogii. In particular, we evaluated the role of UVR in inducing photoinhibition and the potential mitigation of this negative effect by an increase in temperature. Cultures were pre-acclimated at two temperatures - 18°C and 23°C - and exposed to different radiation treatments - UVR+PAR (280-700nm); UV-A+PAR (315-700nm) and PAR only (400-700nm) under two temperatures: 18°C (local surface summer water temperature) and 23°C (simulating a potential increase estimated by the year 2100). Exposure to natural solar radiation resulted in UVR-induced photoinhibition that was significantly higher in T. weissflogii than in C. gracilis. Both species benefited from the higher temperature (23°C) resulting in a lower photoinhibition as compared to samples exposed at 18°C. Inter-specific differences were determined in regard to the heat dissipation processes (NPQ) which were higher at high temperatures, and much more evident in C. gracilis than in T. weissflogii. The analyses of inhibition and recovery rates under different irradiances indicate that the balance between negative (inhibition) and positive (repair-dissipation) effects shifted towards a more positive balance with increasing temperature. Our results highlight for a beneficial effect of temperature on photosynthesis performance during exposure to UVR, although important inter-specific differences are found, probably due to differences in cell size as well as in their distribution within the oceanic realm (i.e., coastal versus oceanic species).
Assuntos
Diatomáceas/efeitos da radiação , Fotossíntese/efeitos da radiação , Raios Ultravioleta , Diatomáceas/enzimologia , Complexo de Proteína do Fotossistema II/metabolismo , Pigmentos Biológicos/metabolismo , Luz Solar , TemperaturaRESUMO
Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.