IAL   21557
INSTITUTO DE AGROBIOTECNOLOGIA DEL LITORAL
Unidad Ejecutora - UE
artículos
Título:
Plastidic Phosphoglycerate Kinase from Phaeodactylum tricornutum: On the critical role of cysteine residues for the enzyme function
Autor/es:
M.B. BOSCO; M. ALEANZI; A.A. IGLESIAS
Revista:
PROTIST
Editorial:
ELSEVIER GMBH
Referencias:
Lugar: Jena; Año: 2012 vol. 163 p. 188 - 203
ISSN:
1434-4610
Resumen:
Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in
photosynthetic organisms, cat-alyzing 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. (C) 2011 Elsevier GmbH. All rights reserved.