Heterologous Expression and Characterization of Phaeodactylum tircornutum Phosphoglycerate Kinase-1

M.B. BOSCO, M. ALEANZI, A.A. IGLESIAS

Villa Carlos Paz, Córdoba, Argentina

Congreso; XLIV Reunión Anual de SAIB; 2008

SAIB

EN-P08.H E T E R O L O G O U S E X P R E S S I O N A N DCHARACTERIZATION OF P. Tr i c o r n u t u m’SPHOSPHOGLYCERATE KINASE-1Bosco MB, Aleanzi M, Iglesias AA.Laboratorio de Enzimología Molecular, FBCB, UNL. Paraje “ElP o z o ” , C C 2 4 2 , S 3 0 0 Z A A S a n t a F e . E - m a i l :mbbosco@fbcb.unl.edu.arDiatoms are key constituents of aquatic phytoplanktoncommunities. Their contribution to marine primary productivitywas estimated between 30-40%. Because of its small genome andthe possibility of being routinely transformed, Phaeodactylumtricornutum has emerged as a model species for dissecting diatombiology. Our interest is to characterize enzymes involved in carbonmetabolism in the diatom. Phosphoglycerate kinase-1 (E.C. 2.7.2.3;PGKase-1), which is a key step in the glycolytic pathway, generatesATP by conversion of 1,3-bis-phospho-glycerate into 3-phosphoglycerate,in a reversible reaction that needs Mg2+ as essential cation.Expression of the gene encoding P. tricornutum PGKase-1 inEscherichia coli rendered a soluble, active and recombinantenzyme. After purification, kinetic, regulatory and structuralproperties of the recombinant enzyme were characterized; and theproperties were compared with those reported for PGKases fromother sources. The specific activity of the purified diatom enzymewas 10.7 U/mg; and it exhibited optima values of temperature (of 30ºC) and pH (of 8.0). Structural characterization was carried outthrough native PAGE and homology modeling, results agreeingwith PGKase-1 being a monomeric enzyme with two possibleconformations: open, in absence of substrates; and closed, when theenzyme is forming a ternary complex with its two substrates.