Architectural Patterns

Abstract

Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C-3 and C-4 metabolites. This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (Pck(Tk)). Pck(Tk) showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp. Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in PckTk. However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs. The recombinant Pck(Tk) actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+. The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the K-m value for oxaloacetate was much lower than that for phosphoenolpyruvate. The transcription and activity levels in T. kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions. These results agreed with the role of Pck(Tk) in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon. Additionally, under gluconeogenic conditions, we observed higher expression levels of Pck(Tk) on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon.