Biochemical and structural characterization of two phosphoenolpyruvate carboxykinases fromthe green alga Chlamydomonas reinhardtii.

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

Mendoza

Congreso; LVIII Reunion SAIB; 2022

SAIB

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. 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 algae Chlamydomonas reinhardtii (Chlre PEPCK).In this work we identified two possible PEPCK isoforms in C. reinhardtii crude extracts, both originated from a unique gene and were named Chlre PEPCK1 and Chlre PEPCK2.We cloned and purified the two enzymes and studied their kinetics, oligomerization forms and response to different metabolites. We assayed the carboxylating and decarboxylating activity of Chlre PEPCK1 and Chlre PEPCK2 to find that the two isoforms are capable of catalyzing both reactions in vitro but they have different decarboxylating parameters, being Chlre PEPCK1 more active than Chlre PEPCK2. The native molecular masses of the Chlre PEPCKs weredetermined by gel filtration chromatography and we observed that ChlreP EPCK1 has an hexameric form while Chlre PEPCK2 is mainly monomeric, implying that Chlre PEPCK2 lacks the needed residues for the monomer-monomer interaction. Last, we studied the influence of different metabolites in the activity of both isoforms. Chlre PEPCK1 activity is affected by the presence of citrate, L-Phe and malate, while Chlre PEPCK2 is regulated by citrate, L-Phe and L-Gln. All in all, it can be concluded that both Chlre PEPCKsare 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 may reflect an in vivo intricate regulation involving allosteric regulation accompanied by oligomeric state changes.