Characterization of a phosphoenolpyruvate carboxykinase from Chlamydomonas reinhardtii

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

Virtual

Congreso; 3er. Encuentro & 1er. Workshop, Red Argentina de Tecnología Enzimática (RedTez).; 2021

RedTez

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme distributed in all groups of organisms. It catalyzes the reversible reaction of decarboxylation and phosphorylation of OAA to generate PEP and CO2 using a nucleotide molecule or PPi for the transfer of phosphoryl, and requires the presence of a divalent metal (generally Mn2+) for its activity. Three groups can be distinguished within the PEPCKs, based strictly on the specificity of the phosphate donor: the ATP-dependent PEPCKs, found mainly in bacteria, yeast, higher plants and trypanosomatids; the GTP-dependent PEPCKs, present in molluscs, insects, fungi and vertebrates and the PPi-dependent PEPCKs found in some bacteria and protozoa. 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. So far, in green microalgae, it has not been described its function or kinetics. In our group, we decided to determine the physiological and biochemical role of a PEPCK from Chlamydomonas reinhardtii (ChlrePEPCK). First, we studied the transcriptional expression of ChlrePEPCK at two different times of the light/dark cycle and found that its relative expression is higher in light condition than in dark condition. We could also detect ChlrePEPCK in C. reinhardtii crude extracts by Western Blot. Moreover, we tested for PEPCK activity in C. reinhardtii crude extracts and we could measure it in both directions, being the velocity of OAA decarboxylation greater than that of PEP carboxylation. Second, we analysed ChlrePEPCK 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. 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: a typical PEPCK folding of a mixed α/β structure that consists of an N-terminal and a C-terminal mononucleotide-binding domain, with the two domains separated by a deep cleft which contains the active site. Third, ChlrePEPCK was cloned and purified to homogeneity and its biochemical properties were characterized. After studying its thermal and pH behaviour, we found that ChlrePEPCK carboxylates PEP and decarboxylates OAA with a hyperbolic response and maximum activity at pH 6 and 7, respectively and at 25°C. Again, PEPCK activity was higher in the direction of OAA decarboxylation. It has been reported that PEPCK activity in plants is affected by several metabolites. So, to survey potential regulatory mechanisms of algae PEPCK, the response of the purified enzyme to several metabolites, including glycolytic and Krebs cycle intermediates and amino acids, were tested. We found that ChlrePEPCK is allosterically modulated by some metabolic intermediates.