Understanding carbon metabolism in green algae: Characterization of Chlamydomonas reinhardtii PEPCK.

TORRESI, FLORENCIA; DIEGO FABIAN GOMEZ CASATI; MARTIN, M.

Virtual

Congreso; Reunión conjunta 2020 SAIB-SAMIGE; 2020

SAIB

Phosphoenolpyruvate carboxykinase EC 4.1.1.32 (GTP-dependent) or EC4.1.1.49 (ATP-dependent) is a widely distributed enzyme which catalyses thereversible decarboxylation and phosphorylation of oxaloacetate (OAA) to yieldphosphoenolpyruvate (PEP) and carbon dioxide (CO2), using ATP or GTPfor the phosphoryl transfer, and requires a divalent metal ion for activity.PEPCKs can be divided into two groups, based on its strict specificity towardsthe nucleotide substrate: the ATP-dependent family, found in bacteria, yeast,higher plants and trypanosomatids; and the GTP-dependent family, found inmolluscs, fungi, insects and vertebrates. The primary role of PEPCK in mostorganisms is the formation of PEP in the first committed step ofgluconeogenesis. In leaves of Crassulacean acid metabolism (CAM) and C4 plants aswell as in some diatoms, PEPCK functions as a decarboxylase involved in CO2-concentratingmechanisms.Since neither PEPCK function nor kinetic or regulation properties havebeen described in green microalgae, we decided to determine the physiologicaland biochemical role of PEPCK from Chlamydomonasreinhardtii (ChlrePEPCK). To thisend, we analysed its sequence by comparison with homologous from other algaeand plants: the four domains described for PEPCKs (PCK domain, kinase-1adomain, kinase 2 domain and ATP-binding motif) were highly conserved in ChlrePEPCK. Moreover, we built ahomology model of ChlrePEPCK usingthe 3D structure of T. cruzi PEPCK(PDB code 1II2) as template finding that ChlrePEPCK model exhibits a fold similarto 1II2, with somedifferences in the ATP-binding motif. ChlrePEPCK was also cloned and purified to homogeneity and itsbiochemical properties were characterized. After studying its thermal and pHbehaviour, we found that ChlrePEPCK carboxylatesPEP with a hyperbolic response and maximum activity at pH 6 and 25°C. Our findingsmay contribute to a better understanding of PEPCK evolutionary process in thegreen lineage and to gain knowledge of its role in carbon metabolism.