Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function

Autor: Mabel Cristina Aleanzi, María Belén Bosco, Alberto A. Iglesias
Rok vydání: 2011
Předmět:
Models
Molecular
Nitroprusside
Molecular Sequence Data
PHOSPHOGLYCERATE KINASE
Molecular cloning
medicine.disease_cause
Microbiology
Dithiothreitol
Sulfenic Acids
Ciencias Biológicas
chemistry.chemical_compound
Thioredoxins
medicine
Escherichia coli
Phaeodactylum tricornutum
Amino Acid Sequence
Cysteine
Disulfides
Plastids
Cloning
Molecular
Enzyme Inhibitors
Enzyme Assays
chemistry.chemical_classification
Diamide
Diatoms
Phosphoglycerate kinase
biology
REDOX REGULATION
Glutathione
Hydrogen Peroxide
Bioquímica y Biología Molecular
biology.organism_classification
Recombinant Proteins
Enzyme Activation
Phosphoglycerate Kinase
Enzyme
Biochemistry
chemistry
Mutagenesis
Site-Directed
Electrophoresis
Polyacrylamide Gel
PHAEODACTYLUM TRICORNUTUM
Oxidation-Reduction
Sequence Alignment
CIENCIAS NATURALES Y EXACTAS
Plasmids
Zdroj: Protist. 163(2)
ISSN: 1618-0941
Popis: 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. Fil: Bosco, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Databáze: OpenAIRE
Externí odkaz: