Molecular and biochemical characterization of the sunflower (Helianthus annuus L.) cytosolic and plastidial enolases in relation to seed development

Publication date: July 2018 Source:Plant Science, Volume 272 Author(s): M.A. Troncoso-Ponce, J. Rivoal, S. Dorion, R. Sánchez, M. Venegas-Calerón, A.J. Moreno-Pérez, S. Baud, R. Garcés, E. Martínez-Force In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphogly...
Source: Plant Science - Category: Biology Source Type: research