BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF TWO PHOSPHOENOLPYRUVATE CARBOXYKINASES FROMTHE GREEN ALGA CHLAMYDOMONAS REINHARDTII

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

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2022

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme distributed in all groups of organisms and catalyzes the reversible reaction of decarboxylation and phosphorylation of OAA to generate PEP and CO2 and it plays mainly a gluconeogenic role in green algae. The partitioning of carbon skeletons between PEP and OAA by the action of PEPCK is potentially important in the inter-conversion of sugars, organic acids, amino acids, aromatic compounds and lipids, suggesting that PEPCK must be strictly regulated. Since neither PEPCK function nor kinetic or regulation properties have been described in green microalgae, we decided to study the physiological and biochemical role of the PEPCK from the model green alga Chlamydomonas reinhardtii (ChlrePEPCK).In this work we identified two possible PEPCK isoforms in C. reinhardtii crude extracts, both originated from a unique gene (Cre02.g141400), one of 68 kDa (ChlrePEPCK1) and the other of 62 kDa (ChlrePEPCK2); then, we purified the two enzymes and studied their kinetics, oligomerization forms and response to different metabolites. We assayed the carboxylating and decarboxylating activity of ChlrePEPCK1 and ChlrePEPCK2 to found that the two isoforms are capable of catalyzing both reactions in vitro but they have different decarboxylating parameters, being ChlrePEPCK1 more active than ChlrePEPCK2. The native molecular masses of the ChlrePEPCKs were determined by gel filtration chromatography and we observed that ChlrePEPCK1 has a hexameric form while ChlrePEPCK2 is mainly monomeric, implying that ChlrePEPCK2 lacks the needed residues for the monomer-monomer interaction. Last, we studied the influence of different metabolites in the activity of both isoforms. ChlrePEPCK1 activity is affected by the presence of citrate, L-Phe and malate, while ChlrePEPCK2 is regulated by citrate, L-Phe and L-Gln. All in all, it can be conclude that both ChlrePEPCKs are active in vitro and have different kinetics parameters, oligomeric structures and they also respond differently to the presence of amino acids and Krebs cycle intermediates. The obtained results allow us to gain insight into the green algae biochemical regulation of PEPCK and to contribute to better understand PEPCK evolution and its role in central carbon metabolism.