Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

Aich, Sanjukta; Imabayashi, Fumie; Delbaere, L. T. J.

doi:10.1016/s1046-5928(03)00189-x

Cited by 20 publications

(14 citation statements)

References 27 publications

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“…1B), whose -electron system stacked onto the guanine ring in the human enzyme (3). Nevertheless, the K m value of Pck Tk for GDP (18.5 M) was within levels similar to those of its eucaryal and bacterial counterparts (1,5,14,20). Another aromatic residue at a presently undefined position may compensate for the function of the missing Phe residue, or a different mode of interaction may exist in the nucleotide binding of the archaeon-type enzymes.…”

Section: Discussionmentioning

confidence: 58%

“…As seen in the case of other GTP-PCKs (5,14,20), ITP and IDP acted as alternative nucleotide cofactors with similar V max values and slightly higher K m values than those for GTP and GDP. ATP and ADP were poor cofactors for Pck Tk but gave relatively high V max values (10 to 30% of those with GTP and GDP) compared to those of classical GTP-PCKs (1,14,20,24). However, the K m values for ATP and ADP were much higher than those of their guanyl counterparts.…”

Section: Resultsmentioning

confidence: 90%

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

et al. 2004

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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...

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Section: Discussionmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 90%

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

et al. 2004

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“…The Pyc assay mixture (1 ml) contained 100 mM Tris-HCl pH 7.8, 5 mM MgCl 2 , 50 mM NaHCO 3 , 5 mM sodium pyruvate, 2 mM ATP, 0.1 mM NADH, 2 U porcine malate dehydrogenase (Sigma-Aldrich), and cell extract [50]. The Ppck activity assay mixture (1 ml) contained 100 mM Hepes buffer pH 7.8, 10 mM MgCl 2 , 0.5 mM MnCl 2 , 1 mM DTT, 50 mM NaHCO 3 , 0.25 mM NADH, 2.5 mM phosphoenolpyruvate, 2.5 mM GDP (or ADP), 10 U porcine malate dehydrogenase (SigmaAldrich), and cell extract [31].…”

Section: Methodsmentioning

confidence: 99%

“…The mutant in which GSU3385 was deleted, designated PPCK1, lacked the Ppck enzyme activity ( Table 2), demonstrating that the GSU3385 gene codes for this enzyme. When the wild-type was assayed, activity was 10-fold higher with GDP than with ADP, suggesting that the enzyme belongs to the class of monomeric GTP dependent Ppck enzymes [31].…”

Section: Redundancy In the Synthesis Of Pep From Pyruvate: Phosphoenomentioning

confidence: 99%

Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

Segura¹,

Mahadevan²,

Juárez³

et al. 2005

PLoS Comput Biol

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Previous model-based analysis of the metabolic network of Geobacter sulfurreducens suggested the existence of several redundant pathways. Here, we identified eight sets of redundant pathways that included redundancy for the assimilation of acetate, and for the conversion of pyruvate into acetyl-CoA. These equivalent pathways and two other sub-optimal pathways were studied using 5 single-gene deletion mutants in those pathways for the evaluation of the predictive capacity of the model. The growth phenotypes of these mutants were studied under 12 different conditions of electron donor and acceptor availability. The comparison of the model predictions with the resulting experimental phenotypes indicated that pyruvate ferredoxin oxidoreductase is the only activity able to convert pyruvate into acetylCoA. However, the results and the modeling showed that the two acetate activation pathways present are not only active, but needed due to the additional role of the acetyl-CoA transferase in the TCA cycle, probably reflecting the adaptation of these bacteria to acetate utilization. In other cases, the data reconciliation suggested additional capacity constraints that were confirmed with biochemical assays. The results demonstrate the need to experimentally verify the activity of key enzymes when developing in silico models of microbial physiology based on sequence-based reconstruction of metabolic networks.

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“…While microbial enzymes often use ATP as a phosphate donor, the C. glutamicum PEP carboxykinase has been shown to be highly specific for GTP (26)(27)(28)(29) and thus represents a notable exception. The kinetic analysis of the purified enzyme revealed ATP to inhibit PEP carboxykinase activity in the oxaloacetate-forming reaction (28), indicating that the enzyme mainly functions in gluconeogenesis and not in anaplerosis under physiological conditions.…”

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confidence: 99%

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

Klaffl¹,

Brocker²,

Kalinowski³

et al. 2013

Journal of Bacteriology

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c DNA affinity chromatography with the promoter region of the Corynebacterium glutamicum pck gene, encoding phosphoenolpyruvate carboxykinase, led to the isolation of four transcriptional regulators, i.e., RamA, GntR1, GntR2, and IolR. Determination of the phosphoenolpyruvate carboxykinase activity of the ⌬ramA, ⌬gntR1 ⌬gntR2, and ⌬iolR deletion mutants indicated that RamA represses pck during growth on glucose about 2-fold, whereas GntR1, GntR2, and IolR activate pck expression about 2-fold irrespective of whether glucose or acetate served as the carbon source. The DNA binding sites of the four regulators in the pck promoter region were identified and their positions correlated with the predicted functions as repressor or activators. The iolR gene is located upstream and in a divergent orientation with respect to a iol gene cluster, encoding proteins involved in myoinositol uptake and degradation. Comparative DNA microarray analysis of the ⌬iolR mutant and the parental wild-type strain revealed strongly (>100-fold) elevated mRNA levels of the iol genes in the mutant, indicating that the primary function of IolR is the repression of the iol genes. IolR binding sites were identified in the promoter regions of iolC, iolT1, and iolR. IolR therefore is presumably subject to negative autoregulation. A consensus DNA binding motif (5=-KGWCHTRACA-3=) which corresponds well to those of other GntR-type regulators of the HutC family was identified. Taken together, our results disclose a complex regulation of the pck gene in C. glutamicum and identify IolR as an efficient repressor of genes involved in myo-inositol catabolism of this organism.

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Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase

Cited by 20 publications

References 27 publications

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

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

Computational and Experimental Analysis of Redundancy in the Central Metabolism of Geobacter sulfurreducens

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

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