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Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene.

ABSTRACT: The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% identical and 79.2% similar to the E. coli enzyme. The A. succiniciproducens pckA transcription start site was determined, and putative promoter regions were identified. The recombinant enzyme was overexpressed in E. coli. The purified enzyme was indiscernible from the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.

SUBMITTER: Laivenieks M

PROVIDER: S-EPMC168520 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene.

Laivenieks M M Vieille C C Zeikus J G JG

Applied and environmental microbiology 19970601 6

The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% ident ...[more]

PMID: 9172347

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Molecular and functional characterization of the Rhodopseudomonas palustris no. 7 phosphoenolpyruvate carboxykinase gene.

Project description:The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacterium Rhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of the pckA initiation codon and an upstream sequence similar to sigma70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, the pckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.

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Neddylation of phosphoenolpyruvate carboxykinase 1 controls glucose metabolism.

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Regulation of the acuF gene, encoding phosphoenolpyruvate carboxykinase in the filamentous fungus Aspergillus nidulans.

Project description:Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.

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Function of phosphoenolpyruvate carboxykinase in mammary gland epithelial cells.

Project description:Previously, we have shown that Pck1 expression in mammary gland adipocytes and white adipose tissue maintains triglyceride stores through glyceroneogenesis, and these lipids were used for synthesis of milk triglycerides during lactation. Reduced milk triglycerides during lactation resulted in patterning of the newborn for insulin resistance. In this study, the role of Pck1 in mammary gland epithelial cells was analyzed. The developmental expression of Pck1 decreased in isolated mouse mammary gland epithelial cells through development and during lactation. Using HC11, a clonal mammary epithelial cell line, we found that both Janus kinase 2 signal transducers and activators of transcription 5 and the AKT pathways contributed to the repression of Pck1 mRNA by prolactin. These pathways necessitate three accessory factor regions of the Pck1 promoter for repression by prolactin. Using [U-(13)C(6)]glucose, [U-(13)C(3)]pyruvate, and [U-(13)C(3)]glycerol in HC11 cells, we determined that Pck1 functions in the pathway for the conversion of gluconeogenic precursors to glucose and contributes to glycerol-3-phosphate synthesis through glyceroneogenesis. Therefore, Pck1 plays an important role in both the mammary gland adipocytes and epithelial cells during lactation.

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Structural insights into the mechanism of phosphoenolpyruvate carboxykinase catalysis.

Project description: Not available

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Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase.

Project description:The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7 s-1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.

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