First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Abstract

Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO 2 , 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 Pck Tk . 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 Mn 2؉ and Mg 2؉ . 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.Recent progress in the research on hyperthermophilic archaea has clarified the presence of unique glycolytic and gluconeogenic pathways distinct from those in mesophilic organisms. In Thermococcus and Pyrococcus spp. belonging to the order Thermococcales in Euryarchaeota, glucose is metabolized by a modified Embden-Meyerhof pathway including ADP-dependent kinases and glyceraldehyde 3-phosphate:ferredoxin oxidoreductase (21, 25). After pyruvate is formed through glycolysis, the terminal reactions of oxidative glucose degradation are the conversion of pyruvate to acetate and CO 2 as end products. Pyruvate:ferredoxin oxidoreductase oxidizes pyruvate to acetyl coenzyme A (acetyl-CoA), and acetyl-CoA synthetase (ADP forming) catalyzes acetate formation from acetyl-CoA, coupled with the phosphorylation of ADP (7, 21). In the direction of gluconeogenesis, phosphoenolpyruvate (PEP) synthase is thought to function in the supply of PEP from pyruvate. Although a wealth of knowledge about these pathways has accumulated, there is still very little information on how these metabolites are converted to C 4 compounds, and vice versa, in hyperthermop...

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7

“…The absorbance change at 340 nm was monitored for 10 min. Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) activity was determined under aerobic conditions by the method of Fukuda et al (2004) using 50 mM NaHCO 3 , 5 mM MnCl 2 , 10 mM phosphoenolpyruvate (PEP), and 2 mM GDP in 100 mM imidazole buffer (pH 7.2). The reaction was started by addition of protein and incubated at 37 uC for 30 min, stopped by addition of 50 mM EDTA and cooled to 4 uC.…”

Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

“…There was no obvious difference in the signal intensities under all conditions examined, indicating constitutive transcription without regulation depending on the carbon source. This profile is significantly different from that of Tk-pck, of which transcription was induced under gluconeogenic conditions (Fukuda et al 2004). We further noticed that the transcription of Tk-mae appeared to be much weaker than that of Tk-pck.…”

“…The genome analysis also revealed that the TCA cycle is incomplete because of the absence of enzymes for many reactions. We have recently reported the first characterization and expression profile of an archaeal PCK from T. kodakaraensis (Tk-Pck) (Fukuda et al 2004). The characteristics of the recombinant protein and expression profile of Tk-Pck were consistent with a role in providing PEP from OAA as the first step of gluconeogenesis.…”

“…This enzyme could be involved in C4 and C3 interconversions in this archaeon, following the previous report that PEP carboxykinase links OAA and PEP (Fukuda et al 2004). The malic enzyme examined here was a homodimeric enzyme preferentially utilizing NADP(H) as a cofactor.…”

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

“…ATP-PEPCKs are mainly present in bacteria, yeast, and plants, whereas GTP-PEPCKs are mostly present in higher eukaryotes, archaea, and some bacteria (21). PEP carboxylase exists in various bacteria and in limited archaea and eukaryotes.…”