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Applied and Environmental Microbiology Aug 2013Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase...
Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.
Topics: Escherichia coli; Industrial Microbiology; Metabolic Engineering; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (ATP); Phosphoenolpyruvate Carboxylase; Recombinant Proteins; Succinic Acid
PubMed: 23747698
DOI: 10.1128/AEM.00826-13 -
Proceedings of the National Academy of... Aug 2021(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic...
(Mtb) infection is difficult to treat because Mtb spends the majority of its life cycle in a nonreplicating (NR) state. Since NR Mtb is highly tolerant to antibiotic effects and can mutate to become drug resistant (DR), our conventional tuberculosis (TB) treatment is not effective. Thus, a novel strategy to kill NR Mtb is required. Accumulating evidence has shown that repetitive exposure to sublethal doses of antibiotics enhances the level of drug tolerance, implying that NR Mtb is formed by adaptive metabolic remodeling. As such, metabolic modulation strategies to block the metabolic remodeling needed to form NR Mtb have emerged as new therapeutic options. Here, we modeled in vitro NR Mtb using hypoxia, applied isotope metabolomics, and revealed that phosphoenolpyruvate (PEP) is nearly completely depleted in NR Mtb. This near loss of PEP reduces PEP-carbon flux toward multiple pathways essential for replication and drug sensitivity. Inversely, supplementing with PEP restored the carbon flux and the activities of the foregoing pathways, resulting in growth and heightened drug susceptibility of NR Mtb, which ultimately prevented the development of DR. Taken together, PEP depletion in NR Mtb is associated with the acquisition of drug tolerance and subsequent emergence of DR, demonstrating that PEP treatment is a possible metabolic modulation strategy to resensitize NR Mtb to conventional TB treatment and prevent the emergence of DR.
Topics: Antitubercular Agents; Drug Resistance, Microbial; Drug Tolerance; Humans; Hypoxia; Mycobacterium tuberculosis; Phosphoenolpyruvate; Tuberculosis
PubMed: 34426499
DOI: 10.1073/pnas.2105800118 -
Biophysical Journal May 2010Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier...
Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg(2+) binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg(2+) binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-(13)C(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO(3)(2-) stretching vibrations); and 967 cm(-1) (symmetric PO(3)(2-) stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm(-1). The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na(+) exhibited the same band positions as in the presence of K(+), indicating very similar interaction strengths between ligand and protein in both cases.
Topics: Absorption; Animals; Biocatalysis; Enzyme Activation; Magnesium; Phosphoenolpyruvate; Protein Binding; Protein Conformation; Pyruvate Kinase; Rabbits; Sodium; Spectrophotometry, Infrared
PubMed: 20441757
DOI: 10.1016/j.bpj.2009.12.4335 -
Journal of Advanced Research Dec 2022Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis.
INTRODUCTION
Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis.
OBJECTIVES
This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus.
METHODS
We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function.
RESULTS
The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%.
CONCLUSION
Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.
Topics: Brassica napus; Gene Expression Regulation, Plant; Phosphoenolpyruvate; Plant Oils; Seeds; Chloroplasts
PubMed: 35907629
DOI: 10.1016/j.jare.2022.07.008 -
FEMS Microbiology Reviews Sep 2005In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that... (Review)
Review
In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.
Topics: Bacteria; Gene Expression Regulation, Bacterial; Glucose; Oxaloacetates; Pentose Phosphate Pathway; Phosphoenolpyruvate; Pyruvic Acid
PubMed: 16102602
DOI: 10.1016/j.femsre.2004.11.002 -
Scientific Reports Nov 2021The deazaflavin cofactor F is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy,...
The deazaflavin cofactor F is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F biosynthesis to optimize F production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space-time yield of F compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F-production system and will allow the recombinant in vivo use of F-dependent enzymes for biocatalysis and protein engineering applications.
Topics: Escherichia coli; Glyceric Acids; Phosphoenolpyruvate; Phosphotransferases (Paired Acceptors); Polyglutamic Acid; Riboflavin
PubMed: 34741069
DOI: 10.1038/s41598-021-01224-3 -
Molecular Microbiology Apr 2006The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but...
The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax. The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli, and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax, which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.
Topics: Amino Acid Sequence; Archaeal Proteins; Carbohydrate Metabolism; Catalysis; Cloning, Molecular; Genes, Archaeal; Molecular Sequence Data; Phosphoenolpyruvate; Phosphotransferases (Paired Acceptors); Pyruvate, Orthophosphate Dikinase; Pyruvic Acid; Thermoproteus
PubMed: 16573681
DOI: 10.1111/j.1365-2958.2006.05098.x -
Journal of Bacteriology Apr 2015Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl...
UNLABELLED
Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and Pi) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13: 657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains.
IMPORTANCE
Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains.
Topics: Adenosine Monophosphate; Adenosine Triphosphate; Aldehyde-Lyases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Glyoxylates; Hydrogen-Ion Concentration; Lactobacillus plantarum; Oxaloacetic Acid; Pentosephosphates; Phosphoenolpyruvate
PubMed: 25605308
DOI: 10.1128/JB.02380-14 -
Cancer Medicine Jan 2023Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential...
BACKGROUND
Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer.
METHODS
We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer.
RESULTS
PEPCK-M was highly expressed in estrogen receptor-positive (ER ) breast cancers. Decreased cell proliferation and G /G arrest were induced in ER breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER breast cancer were attenuated by PCK2 knockdown.
CONCLUSION
PEPCK-M promotes proliferation and cell cycle progression in ER breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER breast cancer.
Topics: Humans; Female; Phosphoenolpyruvate; Receptors, Estrogen; Breast Neoplasms; Glutamine; Phosphoenolpyruvate Carboxykinase (ATP); Mitochondria; TOR Serine-Threonine Kinases; Mechanistic Target of Rapamycin Complex 1
PubMed: 35757841
DOI: 10.1002/cam4.4969 -
The Journal of Biological Chemistry Jul 2023Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance....
Glycolysis is the primary metabolic pathway in the strictly fermentative Streptococcus pneumoniae, which is a major human pathogen associated with antibiotic resistance. Pyruvate kinase (PYK) is the last enzyme in this pathway that catalyzes the production of pyruvate from phosphoenolpyruvate (PEP) and plays a crucial role in controlling carbon flux; however, while S. pneumoniae PYK (SpPYK) is indispensable for growth, surprisingly little is known about its functional properties. Here, we report that compromising mutations in SpPYK confers resistance to the antibiotic fosfomycin, which inhibits the peptidoglycan synthesis enzyme MurA, implying a direct link between PYK and cell wall biogenesis. The crystal structures of SpPYK in the apo and ligand-bound states reveal key interactions that contribute to its conformational change as well as residues responsible for the recognition of PEP and the allosteric activator fructose 1,6-bisphosphate (FBP). Strikingly, FBP binding was observed at a location distinct from previously reported PYK effector binding sites. Furthermore, we show that SpPYK could be engineered to become more responsive to glucose 6-phosphate instead of FBP by sequence and structure-guided mutagenesis of the effector binding site. Together, our work sheds light on the regulatory mechanism of SpPYK and lays the groundwork for antibiotic development that targets this essential enzyme.
Topics: Humans; Anti-Bacterial Agents; Fosfomycin; Kinetics; Phosphoenolpyruvate; Pyruvate Kinase; Streptococcus pneumoniae; Drug Resistance, Bacterial
PubMed: 37286036
DOI: 10.1016/j.jbc.2023.104892