UNDERSTANDING CARBON METABOLISM IN GREEN ALGAE: CHARACTERISATION OF CHLAMYDOMONAS REINHARDTII PEPCK

MARTÍN, MARIANA; GOMEZ-CASATI, DIEGO F.; TORRESI, FLORENCIA

Congreso; Reunión conjunta SAIB|SAMIGE 2020; 2020

Phosphoenolpyruvate carboxykinase EC 4.1.1.32 (GTP-dependent) or EC 4.1.1.49 (ATP-dependent) is a widely distributed enzyme which catalyses the reversible decarboxylation and phosphorylation of oxaloacetate (OAA) to yield phosphoenolpyruvate (PEP) and carbon dioxide (CO2), using ATP or GTP for the phosphoryl transfer, and requires a divalent metal ion for activity. PEPCKs can be divided into two groups, based on its strict specificity towards the nucleotide substrate: the ATP-dependent family, found in bacteria, yeast, higher plants and trypanosomatids; and the GTP-dependent family, found in molluscs, fungi, insects and vertebrates. The primary role of PEPCK in most organisms is the formation of PEP in the first committed step of gluconeogenesis. In leaves of Crassulacean acid metabolism (CAM) and C4 plants as well as in some diatoms, PEPCK functions as a decarboxylase involved in CO2-concentrating mechanisms.Since neither PEPCK function nor kinetic or regulation properties have been described in green microalgae, we decided to determine the physiological and biochemical role of PEPCK from Chlamydomonas reinhardtii (ChlrePEPCK). To this end, we analysed its sequence by comparison with homologous from other algae and plants: the four domains described for PEPCKs (PCK domain, kinase-1a domain, kinase 2 domain and ATP-binding motif) were highly conserved in ChlrePEPCK. Moreover, we built a homology model of ChlrePEPCK using the 3D structure of T. cruzi PEPCK (PDB code 1II2) as template finding that ChlrePEPCK model exhibits a fold similar to 1II2, with some differences in the ATP-binding motif. ChlrePEPCK was also cloned and purified to homogeneity and its biochemical properties were characterized. After studying its thermal and pH behaviour, we found that ChlrePEPCK carboxylates PEP with a hyperbolic response and maximum activity at pH 6 and 25°C. Our findings may contribute to a better understanding of PEPCK evolutionary process in the green lineage and to gain knowledge of its role in carbon metabolism.