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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 -
Journal of Bacteriology May 2022The type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address...
The type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to , the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between function and mannitol utilization. Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.
Topics: Antitoxins; Bacterial Proteins; Enterococcus faecalis; Gene Expression Regulation, Bacterial; Humans; Mannitol; Operon; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phylogeny; Sugars
PubMed: 35404112
DOI: 10.1128/jb.00047-22 -
The New Phytologist Sep 2022Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across...
Crassulacean acid metabolism (CAM) photosynthesis has evolved repeatedly across the plant tree of life, however our understanding of the genetic convergence across independent origins remains hampered by the lack of comparative studies. Here, we explore gene expression profiles in eight species from the Agavoideae (Asparagaceae) encompassing three independent origins of CAM. Using comparative physiology and transcriptomics, we examined the variable modes of CAM in this subfamily and the changes in gene expression across time of day and between well watered and drought-stressed treatments. We further assessed gene expression and the molecular evolution of genes encoding phosphoenolpyruvate carboxylase (PPC), an enzyme required for primary carbon fixation in CAM. Most time-of-day expression profiles are largely conserved across all eight species and suggest that large perturbations to the central clock are not required for CAM evolution. By contrast, transcriptional response to drought is highly lineage specific. Yucca and Beschorneria have CAM-like expression of PPC2, a copy of PPC that has never been shown to be recruited for CAM in angiosperms. Together the physiological and transcriptomic comparison of closely related C and CAM species reveals similar gene expression profiles, with the notable exception of differential recruitment of carboxylase enzymes for CAM function.
Topics: Asparagaceae; Crassulacean Acid Metabolism; Phosphoenolpyruvate Carboxylase; Photosynthesis; Transcriptome
PubMed: 35596719
DOI: 10.1111/nph.18267 -
FEMS Microbiology Reviews May 2021At the junction between the glycolysis and the tricarboxylic acid cycle-as well as various other metabolic pathways-lies the phosphoenolpyruvate...
At the junction between the glycolysis and the tricarboxylic acid cycle-as well as various other metabolic pathways-lies the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node (PPO-node). These three metabolites form the core of a network involving at least eleven different types of enzymes, each with numerous subtypes. Obviously, no single organism maintains each of these eleven enzymes; instead, different organisms possess different subsets in their PPO-node, which results in a remarkable degree of variation, despite connecting such deeply conserved metabolic pathways as the glycolysis and the tricarboxylic acid cycle. The PPO-node enzymes play a crucial role in cellular energetics, with most of them involved in (de)phosphorylation of nucleotide phosphates, while those responsible for malate conversion are important redox enzymes. Variations in PPO-node therefore reflect the different energetic niches that organisms can occupy. In this review, we give an overview of the biochemistry of these eleven PPO-node enzymes. We attempt to highlight the variation that exists, both in PPO-node compositions, as well as in the roles that the enzymes can have within those different settings, through various recent discoveries in both bacteria and archaea that reveal deviations from canonical functions.
Topics: Archaea; Bacteria; Energy Metabolism; Enzymes; Oxaloacetic Acid; Phosphoenolpyruvate; Pyruvic Acid
PubMed: 33289792
DOI: 10.1093/femsre/fuaa061 -
American Journal of Cancer Research 2023Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a key gluconeogenesis enzyme. Its differential expression is related to kidney renal clear cell carcinoma...
Mitochondrial phosphoenolpyruvate carboxykinase (PCK2) is a key gluconeogenesis enzyme. Its differential expression is related to kidney renal clear cell carcinoma (KIRC) malignancy, possibly by influencing energy metabolism. Therefore, it is possible that PKC2 plays a significant part in the emergence and progression of KIRC. To systematically and comprehensively identify the significance of PCK2 in KIRC, we further studied PCK2 in terms of its relationship to clinical features and various clinical subgroups' prognoses. Moreover, we verified the effect of PCK2 and KIRC cells using experimental methods. PCR and western blotting analyses confirmed PCK2 expression in KIRC cell lines and tissues. As a cell model, we constructed cells that overexpress PCK2. Proliferation was detected by EdU experiments. Scratch tests and transwell assays were used, respectively, to analyze cell migration and invasion. Mass spectrometry detected energy metabolite expression in KIRC cells. The findings revealed that KIRC patients with lower levels of PCK2 expression exhibited shorter progression-free intervals, shorter disease-specific survival, and shorter overall survival. The experimental results showed that compared with 293t, PCK2 was downregulated in three KIRC lines (OSRC-2, 786-O, and A498). Relative to surrounding tissues, PCK2 was downregulated in KIRC. PCK2 overexpression inhibited KIRC cell proliferation, migration, and invasion and upregulated energy metabolite expression. Mass spectrometry revealed that thiamine pyrophosphate, cyclic AMP, beta-D-fructose 6-phosphate, lactate, flavin mononucleotide, NAD, NADP, and D-glucose 6-phosphate were upregulated. PCK2 has the potential to serve as both a diagnostic and prognostic molecular biomarker for KIRC, as well as an independent prognostic risk factor for KIRC. It is hoped that PCK2 will emerge as a therapeutic target for KIRC.
PubMed: 37034209
DOI: No ID Found -
Frontiers in Microbiology 2019P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified,...
P2 grows on different carbohydrates as well as alcohols, peptides and amino acids. Carbohydrates such as D-glucose or D-galactose are degraded via the modified, branched Entner-Doudoroff (ED) pathway whereas growth on peptides requires the Embden-Meyerhof-Parnas (EMP) pathway for gluconeogenesis. As for most hyperthermophilic Archaea an important control point is established at the level of triosephophate conversion, however, the regulation at the level of pyruvate/phosphoenolpyruvate conversion was not tackled so far. Here we describe the cloning, expression, purification and characterization of the pyruvate kinase (PK, SSO0981) and the phosphoenolpyruvate synthetase (PEPS, SSO0883) of . The PK showed only catabolic activity [catalytic efficiency (PEP): 627.95 mMs, 70°C] with phosphoenolpyruvate as substrate and ADP as phosphate acceptor and was allosterically inhibited by ATP and isocitrate ( 0.8 mM). The PEPS was reversible, however, exhibited preferred activity in the gluconeogenic direction [catalytic efficiency (pyruvate): 1.04 mMs, 70°C] and showed some inhibition by AMP and α-ketoglutarate. The gene annotated as PEPS/pyruvate:phosphate dikinase (PPDK) revealed neither PEPS nor PPDK activity. Our studies suggest that the energy charge of the cell as well as the availability of building blocks in the citric acid cycle and the carbon/nitrogen balance plays a major role in the carbon switch. The comparison of regulatory features of well-studied hyperthermophilic Archaea reveals a close link and sophisticated coordination between the respective sugar kinases and the kinetic and regulatory properties of the enzymes at the level of PEP-pyruvate conversion.
PubMed: 31031731
DOI: 10.3389/fmicb.2019.00757 -
Frontiers in Plant Science 2022Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as...
Poaceae is the most prominent monocot family that contains the primary cereal crops wheat, rice, and maize. These cereal species exhibit physiological diversity, such as different photosynthetic systems and environmental stress tolerance. Phosphopyruvate carboxylase (PEPC) in Poaceae is encoded by a small multigene family and plays a central role in C-photosynthesis and dicarboxylic acid metabolism. Here, to better understand the molecular basis of the cereal species diversity, we analyzed the gene family in wheat together with other grass species. We could designate seven plant-type and one bacterial-type grass , ppc1a, ppc1b, ppc2a, ppc2b, ppc3, ppc4, ppcC, and ppc-b, respectively, among which ppc1b is an uncharacterized type of . Evolutionary inference revealed that these were derived from five types of ancient (, , , , and ) in three chromosomal blocks of the ancestral Poaceae genome. C-photosynthetic ( ) had evolved from , which seemed to be arisen by a chromosomal duplication event. We observed that was lost in many species but preserved in Pooideae after natural selection. analysis of cereal RNA-Seq data highlighted the preferential expression of in upper ground organs, selective up-regulation of under osmotic stress conditions, and nitrogen response of . Characterization of wheat showed high levels of gene expression in young leaves, transcriptional responses under nitrogen and abiotic stress, and the presence of a Dof1 binding site, similar to in maize. Our results indicate the evolving status of Poaceae PEPCs and suggest the functional association of -derivatives with adaptation to environmental changes.
PubMed: 35958195
DOI: 10.3389/fpls.2022.905894 -
Journal of Molecular Microbiology and... 2015In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might...
In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might catalyze sugar transport as well as sugar phosphorylation. What we have learned in the 50 years since its discovery is that, in addition to these primary functions, the PTS serves as a complex protein kinase system that regulates a wide variety of transport, metabolic and mutagenic processes as well as the expression of numerous genes. Recent operon- and genome-sequencing projects have revealed novel PTS protein-encoding genes, many of which have yet to be functionally defined. The current picture of the PTS is that of a complex system with ramifications in all aspects of cellular physiology. Moreover, its mosaic evolutionary history is unusual and intriguing. The PTS can be considered to serve many prokaryotes in capacities of communication and coordination, as do the nervous systems of animals.
Topics: Bacteria; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Multigene Family; Operon; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases
PubMed: 26159069
DOI: 10.1159/000381215 -
Proceedings of the National Academy of... May 2020The nitrogen-related phosphotransferase system (PTS) of bv. 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls...
The nitrogen-related phosphotransferase system (PTS) of bv. 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTS constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTS is widely conserved in proteobacteria, highlighting its global importance.
Topics: Bacterial Proteins; Carbon; Citric Acid Cycle; Gene Expression Regulation, Bacterial; Nitrogen; Phosphates; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Promoter Regions, Genetic; Rhizobium leguminosarum
PubMed: 32341157
DOI: 10.1073/pnas.1917471117 -
The Journal of Biological Chemistry May 2018Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular...
Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular mechanisms in the regulation of hepatic gluconeogenesis remain to be fully defined. In this study, using murine hepatocytes and a liver-specific knockout mouse model, we explored the physiological role of nuclear factor Y (NF-Y) in regulating hepatic glucose metabolism and the underlying mechanism. We found that NF-Y targets the gluconeogenesis pathway in the liver. Hepatic NF-Y expression was effectively induced by cAMP, glucagon, and fasting Lentivirus-mediated NF-Y overexpression in Hepa1-6 hepatocytes markedly raised the gluconeogenic gene expression and cellular glucose production compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated gluconeogenic gene expression and glucose production. We also provide evidence indicating that CRE-loxP-mediated, liver-specific NF-YA knockout compromises hepatic glucose production. Mechanistically, luciferase reporter gene assays and ChIP analysis indicated that NF-Y activates transcription of the gluconeogenic genes and , by encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase), respectively, via directly binding to the CCAAT regulatory sequence motif in their promoters. Of note, NF-Y enhanced gluconeogenesis by interacting with cAMP-responsive element-binding protein (CREB). Overall, our results reveal a previously unrecognized physiological function of NF-Y in controlling glucose metabolism by up-regulating the gluconeogenic genes and Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.
Topics: Animals; Binding Sites; CCAAT-Binding Factor; CREB-Binding Protein; CRISPR-Cas Systems; Cell Line; Cyclic AMP; Fasting; Gene Deletion; Gene Expression Regulation; Glucagon; Gluconeogenesis; Glucose; Glucose-6-Phosphatase; Hepatocytes; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphoenolpyruvate Carboxykinase (GTP); Primary Cell Culture; Promoter Regions, Genetic; Protein Binding; Signal Transduction
PubMed: 29530977
DOI: 10.1074/jbc.RA117.000508