Redox Characterization of Phaeodactylum tricornutum’s Chloroplastic Phosphoglycerate Kinase

M.B. BOSCO; M. ALEANZI; A.A. IGLESIAS

San Miguel de Tucumán, Argentina

Congreso; XLV Reunión Anual de SAIB; 2009

SAIB

EN-P10.REDOX CHARACTERIZATION OF Phaeodactylumt r i c o r n u t u m ´ s C H L O R O P L A S I T I CPHOSPHOGLYCERATE KINASEBosco MB; Alenazi M; Iglesias AAInstituto de Agrobiotecnología del Litoral (UNL-CONICET).Paraje “El Pozo” CC 242 S300ZAA Santa Fe. E-mail:mbbosco@fbcb.unl.edu.arDiatoms are key constituents of aquatic phytoplanktoncommunities. Because of its small genome and the possibility ofbeing routinely transformed, Phaeodactylum tricornutum hasemerged as a model species for dissecting diatom biology. Ourinterest is to characterize enzymes involved in carbon metabolismin the diatom. Chloroplastic phosphoglycerate kinase-1 (E.C.2.7.2.3; PGKase-1) catalyses the ATP-dependent phosphorylationof 3-phospho-glycerate to 1,3-bis-phospho-glycerate, in areversible reaction needing Mg2+ as essential cofactor, a key step inthe Calvin cycle. Expression of the gene encoding P. tricornutumPGKase-1 in Escherichia coli rendered a soluble and activeenzyme. After purification, kinetic, regulatory and structuralproperties of the recombinant enzyme were characterized. Thepurified enzyme showed inactivation by thiol-modifying reagents,property observed in some animal enzymes but never described fordiatoms. Inhibition constants were obtained for oxidants such asdiamide, sodium nitroprusside and hydrogen peroxide. Reversionof the oxidation was tested with DTT and E. coli thioredoxin andthe activation constants calculated and compared. P tricornutumPGKase-1 has 6 cystein residues. A homology modelled structureserved to predict those cysteines involved in redox regulation andmutants were constructed in order to elucidate the regulatorymechanism.