Phosphoenolpyruvate Research Articles

The PEP-pyruvate-oxaloacetate node: variation at the heart of metabolism.

ABSTRACTAt 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.

RNA-Seq-Based Transcriptomic Analysis of Saccharopolyspora spinosa Revealed the Critical Function of PEP Phosphonomutase in the Replenishment Pathway.

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-ΔPEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.

Metabolic responses of penaeid shrimp to acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus

Vibrio parahaemolyticus is a causative agent of the acute hepatopancreatic necrosis disease (AHPND) that leads to massive mortalities in penaeid shrimp aquaculture worldwide. Mitigation of the significant economic losses caused by AHPND are hampered by knowledge gaps in the pathogenic mechanism of V. parahaemolyticus infection in shrimp. To provide insights into this infection mechanisms, molecular responses of shrimp exposed to V. parahaemolyticus were measured via a metabolomics approach. A gas chromatography–mass spectrometry (GC–MS) platform was employed to generate metabolite profiles of haemolymph from Penaeus vannamei shrimp challenged with V. parahaemolyticus and control shrimp (not exposed to the pathogen). The results revealed increases of several intermediates in the citric acid (TCA) cycle (cis-aconitic acid, citric acid, fumaric acid, isocitric acid and succinic acid) and phosphoenolpyruvic acid (PEP), as well as decreases of six amino acids (threonine, asparagine, 4-aminobutyric acid, histidine, ornithine, glutamine) in challenged shrimp compared to controls. A pathway analysis identified 11 significantly changed pathways due to V. parahaemolyticus infection, which were mostly amino acid metabolisms, TCA cycle and gluconeogenesis. The results suggest an increase in metabolic rate and elevated TCA cycle and gluconeogenesis activities in Vibrio-challenged shrimp due to the high energy demand of the associated immune response. The study provides important new findings about the pathogenic mechanisms of V. parahaemolyticus invasion in shrimp at the metabolic level. Further metabolomics investigations may lead for identification of metabolite biomarkers of V. parahaemolyticus infection for early detection of AHPND.

P32 La semilla de Salvia hispanica L. (chía) promueve la reducción de la grasa corporal y mejora desórdenes del metabolismo lipídico en el tejido adiposo blanco de ratas alimentadas con dieta rica en sacarosa

Introducción: en los últimos años varios estudios se centraron en los mecanismos involucrados en la acumulación de grasa visceral y su rol en la fisiopatología del síndrome metabólico, con el fin de encontrar estrategias terapéuticas para su prevención y/o tratamiento. En este sentido, la semilla de Salvia hispanica L. (chía) ha generado un gran interés como fuente de compuestos bioactivos (ácido alfa-linolénico, fibras, proteínas de alta calidad, vitaminas, minerales y antioxidantes).Objetivos: analizar los efectos de la semilla de chía sobre las vías metabólicas que juegan un papel clave en el manejo de los lípidos en el tejido adiposo blanco y que podrían estar involucrados en la reducción de la adiposidad visceral desarrollada en ratas alimentadas con una dieta rica en sacarosa (DRS).Materiales y métodos: ratas macho Wistar fueron alimentadas con una dieta de referencia (DR) -6 meses- o una DRS -3 meses. Luego, el último grupo se dividió aleatoriamente en dos subgrupos. Un subgrupo continuó recibiendo la DRS hasta los 6 meses y el otro recibió una DRS donde se incorporó la semilla de chía entera como fuente de grasa durante los próximos 3 meses (DRS+CHIA). Al final del período experimental, se analizó en tejido adiposo epididimal: las actividades de enzimas lipogénicas (ATP citrato Liasa ATPCL, sintasa de ácidos grasos FAS, fosfoenolpiruvatocarboxiquinasa PEPCK, enzima malica EM, acetil CoAcarboxilasa ACC) y oxidativas (carnitinapalmitoiltransferasa CPT), los niveles de masa proteica de FAT/CD 36, SREBP-1, AMPK, AMPKp y PKCbeta. Además, se determinaron parámetros antropométricos, ingesta de energía, composición de la carcasa, lípidos plasmáticos, adiposidad visceral y contenido de lípidos en adipocitos. Clampeuglucémica-hiperinsulinémica.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis and Increased Reliance on Gluconeogenesis in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and chemotherapy resistance. Devimistat (CPI-613®) is a novel agent that inhibits two key tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase complexes (KGDH). Our lab has shown that genetic and pharmacologic inhibition of PDH sensitizes cells to chemotherapy and recent clinical trials of chemotherapy plus devimistat have been promising. However, the means by which AML cells adapt to PDH inhibition during devimistat treatment is unknown. Methods: Murine AML cells were used to knockout PDH through the CRISPR-CAS9 system. Viability assays were done using EZQuant or Cell-Titer Glo assays. Glycolytic flux was assessed by measurement of the extracellular acidification rate (ECAR) using the XF24 Extracellular Flux Analyzer. Glucose uptake was measured by Glucose Uptake-Glo assay. Protein levels were assessed by Western blot. Gene expression was measured by qPCR. Amino acid dependencies were assessed by incubating cells in Hank's Balanced Salt Solution with devimistat and either asparagine or glutamate for 4 hours followed by return to complete media and viability assayed after 72-hours. Results: Previous studies have shown that glycolysis can compensate for mitochondrial inhibition so we determined glycolytic rates during PDH inhibition. We have shown previously that genetic PDH loss decreases ECAR levels consistent with decreased glycolytic flux. Devimisat treatment also decreased ECAR levels in K562 and OCI cells in a dose dependent manner. We next examined glucose uptake. PDH knockout cells showed decreased glucose uptake compared to control. This could be due to reduced glucose import or reduced glucose retention. The expression of glucose importer GLUT1 was significantly decreased in cells with genetic or pharmacologic inhibition of PDH. LDHA expression was also reduced in PDH KO cells and devimistat treated cells, contributing to the ECAR decrease. Hexokinase II (HKII) traps glucose in AML cells and docks at the mitochondrial membrane. It can be dislodged with mitochondrial stress and subsequently degraded via the proteasome. PDH knockout cells and devimistat treated cells demonstrated reduced levels of HKII that could be rescued with the proteasomal inhibitor bortezomib. Taken together this data shows that genetic and pharmacologic inhibition of PDH decreases glycolysis by decreasing glucose import and retention. To assess the necessity of the diminished glycolytic activity we treated PDH knockout and devimistat treated cells with the glycolytic inhibitor 2-deoxy-D-glucose. Both devimistat treated and PDH KO cells were exquisitely sensitive to glycolysis inhibition. We next assessed the importance of gluconeogenic amino acids. We specifically looked at glutamate and asparagine as they have been shown to be highly utilized in cancers. Asparagine but not glutamate rescued AML cells from devimistat treatment. Asparaginase which catalysis the conversion of asparagine to aspartic acid, thus limiting the amount of asparagine in the cells sensitizes cells to both genetic and pharmacologic PDH inhibition. Asparagine can be converted into oxaloacetate which can be converted into phosphoenolpyruvate (PEP) for gluconeogenesis. Phosphoenolpyruvate carboxykinase (PCK2) catalyzes the rate limiting step in gluconeogenesis converting oxaloacetate to PEP which can travel backwards through glycolysis. We found that PCK2 was highly upregulated in both the PDH knock cells and devimisat treated cells. Indicating that with the reduced glucose import and retention the cells rely on gluconeogenesis from amino acids to maintain needed glycolytic intermediates. Conclusions: This data shows that PDH inhibition leads to diminished glycolytic rate, increased reliance on amino acid metabolism and increased gluconeogenesis. This in turn suggests that glycolysis inhibition and amino acid deprivation in combination with devminisat mediated PDH inhibition should be explored in clinical trials. Disclosures Pardee: Amgen: Honoraria, Speakers Bureau; Pharmacyclics: Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Rafael: Research Funding; Karyopharm: Research Funding; BMS: Consultancy, Honoraria, Speakers Bureau; Genentech, Inc.: Consultancy; AbbVie: Consultancy; Rafael Pharmaceuticals: Consultancy. OffLabel Disclosure: Devimistat for the treatment of AML

Molecular cloning and expression of hosphoenolpyruvate carboxykinase dominant epitope in Leishmania infantum

Objective Based on the (phosphoenolpyruvate carboxykinase, PEPCK) dominant epitopes gene of Leishmania infantum that has been analyzed and selected, we construct the corresponding recombinant prokaryotic expression vector to obtain the recombinant protein, construct the corresponding recombinant eukaryotic expression vector and verify the expression of eukaryotic vector in NIH3T3 cells, which laid the foundation for the subsequent vaccination and infection of animal experiment. Methods Based on the PEPCK dominant epitopes gene sequence, the recombinant prokaryotic and eukaryotic expression plasmid pET32a- PEPCK and pVAX1- EPEPCK were constructed by PCR reaction and enzyme reaction and were respectively transfected into E. coli and NIH3T3 cells of mice for expression. The expression of prokaryotic recombinant plasmid and the prokaryotic expressed protein purified by HisTrap affinity column were detected through SDS-PAGE and Western-Blot, and the expression of pVAX1-E PEPCK was verified by immunofluorescence. Results The correct sequencing results of the recombinant prokaryotic and eukaryotic vectors showed that the construction of recombinant vectors was successful. The positive results of 48.08 kD of recombinant protein were detected in SDS-PAGE and Western Blot, and immunofluorescence results of NIH3T3 cells transfected with eukaryotic recombinant vectors showed positive results. Conclusion We successfully constructed the recombinant prokaryotic and eukaryotic expression vectors: pET32a- PEPCK and pVAX1-E PEPCK , successfully got the recombinant proteins that followed by proteinic purification and confirmed the expression of pVAX1-E PEPCK in NIH3T3 cells, which is the basis for experiments where animals are vaccinated with a DNA prime-protein boost vaccines. 摘要：目的 基于前期分析并选取的婴儿利什曼原虫 PEPCK 的优势表位基因, 构建相应重组原核表达载体以获得 重组蛋白, 构建相应重组真核表达载体并验证真核载体在 NIH3T3 细胞中的表达, 为后续动物的免疫和感染实验备下 基础。 方法 根据 PEPCK 优势表位基因序列, 经 PCR 反应及酶切连接构建重组原核与真核表达质粒 pET32a- PEPCK 和 pVAX1-E PEPCK 并分别转染至 E. coli 和 NIH3T3 细胞中进行表达。采用 SDS-PAGE 电泳和 Western Blot 鉴定原核重 组质粒在大肠杆菌中的表达和原核表达蛋白被镍柱纯化后的情况, 采用免疫荧光实验验证真核重组质粒在细胞中的 表达。 结果 重组原核与真核载体的正确测序结果表明重组载体构建成功。SDS-PAGE 电泳和 Western Blot 的结果 显示, 大肠杆菌中表达的原核重组蛋白以及纯化后的蛋白在 48.08 kD 处存在条带, 转染了真核重组载体的 NIH3T3 细胞 的免疫荧光结果呈阳性。 结论 成功构建了 PEPCK 优势表位基因的重组原核和真核表达载体: pET32a- PEPCK 和 pVAX1-E PEPCK , 成功表达相应的重组蛋白并纯化并验证了 pVAX1-E PEPCK 在 NIH3T3 细胞中的表达, 为后续 DNA 疫苗初次免疫和蛋白疫苗加强免疫的动物实验备下基础。

Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway

Pancreatic β cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of β cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of β cell function.

Multi-Tissue Acceleration of the Mitochondrial Phosphoenolpyruvate Cycle Improves Whole-Body Metabolic Health

The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2-/- mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator invivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.

In silico Determination of Some Conditions Leading to Glycolytic Oscillations and Their Interference With Some Other Processes in E. coli Cells.

Autonomous oscillations of species levels in the glycolysis express the self-control of this essential cellular pathway belonging to the central carbon metabolism (CCM), and this phenomenon takes place in a large number of bacteria. Oscillations of glycolytic intermediates in living cells occur according to the environmental conditions and to the cell characteristics, especially the adenosine triphosphate (ATP) recovery system. Determining the conditions that lead to the occurrence and maintenance of the glycolytic oscillations can present immediate practical applications. Such a model-based analysis allows in silico (model-based) design of genetically modified microorganisms (GMO) with certain characteristics of interest for the biosynthesis industry, medicine, etc. Based on our kinetic model validated in previous works, this paper aims to in silico identify operating parameters and cell factors leading to the occurrence of stable glycolytic oscillations in the Escherichia coli cells. As long as most of the glycolytic intermediates are involved in various cellular metabolic pathways belonging to the CCM, evaluation of the dynamics and average level of its intermediates is of high importance for further applicative analyses. As an example, by using a lumped kinetic model for tryptophan (TRP) synthesis from literature, and its own kinetic model for the oscillatory glycolysis, this paper highlights the influence of glycolytic oscillations on the oscillatory TRP synthesis through the PEP (phosphoenolpyruvate) glycolytic node shared by the two oscillatory processes. The numerical analysis allows further TRP production maximization in a fed-batch bioreactor (FBR).

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

SummaryIntroduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two‐cell metabolic prototype for an NADP‐malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP‐malate dehydrogenase, pyruvate orthophosphate dikinase and NADP‐malic enzyme from Zea mays, driven by cell‐preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13CO2 labelling demonstrated a 10‐fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP‐malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

Elongation factor P is required for EIIGlc translation in Corynebacterium glutamicum due to an essential polyproline motif.

Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EIIGlc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EIIGlc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EIIGlc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EIIGlc , which has implications for future strain engineering strategies.

Hysteresis of pyruvate phosphate dikinase from Trypanosoma cruzi.

Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.

A Partial C4 Photosynthetic Biochemical Pathway in Rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.

Effects and mechanisms of fatty acid metabolism‑mediated glycolysis regulated by betulinic acid‑loaded nanoliposomes in colorectal cancer.

Although previous studies have demonstrated that triterpenoids, such as betulinic acid (BA), can inhibit tumor cell growth, their potential targets in colorectal cancer (CRC) metabolism have not been systematically investigated. In the present study, BA-loaded nanoliposomes (BA-NLs) were prepared, and their effects on CRC cell lines were evaluated. The aim of the present study was to determine the anticancer mechanisms of action of BA-NLs in fatty acid metabolism-mediated glycolysis, and investigate the role of key targets, such as acyl-CoA synthetase (ACSL), carnitine palmitoyltransferase (CPT) and acetyl CoA, in promoting glycolysis, which is activated by inducing hexokinase (HK), phosphofructokinase-1 (PFK-1), phosphoenolpyruvate (PEP) and pyruvate kinase (PK) expression. The results demonstrated that BA-NLs significantly suppressed the proliferation and glucose uptake of CRC cells by regulating potential glycolysis and fatty acid metabolism targets and pathways, which forms the basis of the anti-CRC function of BA-NLs. Moreover, the effects of BA-NLs were further validated by demonstrating that the key targets of HK2, PFK-1, PEP and PK isoenzyme M2 (PKM2) in glycolysis, and of ACSL1, CPT1a and PEP in fatty acid metabolism, were blocked by BA-NLs, which play key roles in the inhibition of glycolysis and fatty acid-mediated production of pyruvate and lactate. The results of the present study may provide a deeper understanding supporting the hypothesis that liposomal BA may regulate alternative metabolic pathways implicated in CRC adjuvant therapy.

Exploring Amino Sugar and Phosphoenolpyruvate Metabolism to Improve Escherichia coli N-Acetylneuraminic Acid Production.

N-acetyl-d-neuraminic acid (NeuAc) has attracted considerable attention because of its wide-ranging applications. The use of cheap carbon sources such as glucose without the addition of any precursor in microbial NeuAc production has many advantages. In this study, improved NeuAc production was attained through the optimization of amino sugar metabolism pathway kinetics and reservation of a phosphoenolpyruvate (PEP) pool in Escherichia coli. N-acylglucosamine 2-epimerase and N-acetylneuraminate synthase from different sources and their best combinations were used to obtain optimized enzyme kinetics and expression intensity, which resulted in a significant increase in NeuAc production. Next, after a design was engineered for enabling the PEP metabolic pathway to retain the PEP pool, the production of NeuAc reached 16.7 g/L, which is the highest NeuAc production rate that has been reported from using glucose as the sole carbon source.

Targeting shikimate pathway: In silico analysis of phosphoenolpyruvate derivatives as inhibitors of EPSP synthase and DAHP synthase

The shikimate pathway consists of seven enzymatic steps involved in the conversion of erythrose-4-phosphate and phosphoenolpyruvate to chorismate and also responsible to the production of aromatic amino acids, such as phenylalanine, tyrosine, and tryptophan which are essential to the bacterial metabolism. The 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalyze important steps in the shikimate pathway using as substrate the phosphoenolpyruvate (PEP). Due to the importance of PEP in shikimate pathway, its structure has been investigated to develop new bioinspired competitive inhibitors against DAHPS and EPSPS. In the present study, we perform a literature survey of 28 PEP derivatives, then we analyzed the selectivity and affinity of these compounds against the EPSPS and DAHPS structures using consensual molecular docking, pharmacophore prediction, molecular dynamics (MD) simulations, and binding free energy calculations. Here, we propose consistent binding modes of the selected ligands and indicate that their structures show interesting pharmacophoric properties related to multi-targets inhibitors for both enzymes. Our computational results are supported by previous experimental findings related to the interactions of PEP derivatives with DAHPS and EPSPS structures.

Difference in pyruvic acid metabolism between neonatal and adult mouse lungs exposed to hyperoxia.

Neonatal lungs are more tolerant to hyperoxic injury than are adult lungs. This study investigated differences in the response to hyperoxic exposure between neonatal and adult mouse lungs using metabolomics analysis with capillary electrophoresis time-of-flight mass spectrometry (CE- TOFMS). Neonatal and adult mice were exposed to 21% or 95% O2 for four days. Subsequently, lung tissue samples were collected and analyzed by CE-TOFMS. Pyruvate dehydrogenase (PDH) enzyme activity was determined using a microplate assay kit. PDH kinase (Pdk) 1, Pdk2, Pdk3, and Pdk4 mRNA expression levels were determined using quantitative reverse transcription-polymerase chain reaction. Pdk4 protein expression was quantified by Western blotting and Pdk4 protein localization was evaluated by immunohistochemistry. Levels of 3-phosphoglyceric acid, 2-phosphoglyceric acid, phosphoenolpyruvic acid, and lactic acid were significantly elevated in the lungs of hyperoxia-exposed versus normoxia-exposed adult mice, whereas no significant differences were observed with hyperoxia exposure in neonatal mice. PDH activity was reduced in the lungs of adult mice only. Pdk4 mRNA expression levels after hyperoxic exposure were significantly elevated in adult mice compared with that in neonatal mice. Conversely, gene expression levels of Pdk1, Pdk2, and Pdk3 did not differ after hyperoxic exposure in either neonatal or adult mice. Pdk4 protein levels were also significantly increased in adult mouse lungs exposed to hyperoxia and were localized mainly to the epithelium of terminal bronchiole. Specific metabolites associated with glycolysis and gluconeogenesis were altered after hyperoxia exposure in the lungs of adult mice, but not in neonates, which was likely a result of reduced PDH activity due to Pdk4 mRNA upregulation under hyperoxia.

Plasma metabolomic study in perinatally HIV-infected children using 1H NMR spectroscopy reveals perturbed metabolites that sustain during therapy.

BackgroundPerinatally HIV-infected children on anti-retroviral treatment (ART) are reported to have metabolic abnormalities such as dyslipidemia, lipodystrophy, and insulin resistance which potentially increase the risk of diabetes, kidney, liver and cardiovascular disease.ObjectiveTo elucidate HIV-mediated metabolic complications that sustain even during ART in perinatally HIV-infected children.MethodWe have carried out metabolic profiling of the plasma of treatment-naïve and ART-suppressed perinatally HIV-infected children and uninfected controls using 1H nuclear magnetic resonance (NMR) spectroscopy followed by statistical analysis and annotation.ResultValidated multivariate analysis showed clear distinction among our study groups. Our results showed elevated levels of lactate, glucose, phosphoenolpyruvic acid, propionic acid, 2-ketobutyric acid and tricarboxylic acid (TCA) cycle metabolites in untreated HIV-infected children compared to uninfected controls. ART normalized the levels of several metabolites, however the level of lactate, phosphoenolpyruvic acid, oxoglutaric acid, oxaloacetic acid, myoinositol and glutamine remained upregulated despite ART in HIV-infected children. Pathway analysis revealed perturbed propanoate metabolism, amino acid metabolism, glycolysis and TCA cycle in untreated and ART-suppressed HIV-infected children.ConclusionDeveloping therapeutic strategies targeting metabolic abnormalities may be beneficial for preventing diabetes, cardiovascular disease or other associated complications in perinatally HIV-infected children.

Rapidly evolving phosphoenolpyruvate carboxylase Gmppc1 and Gmppc7 are highly expressed in the external seed coat of immature soybean seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a carbon fixation enzyme which probably plays crucial roles in seed development. A greater number of PEPC isoforms are encoded in the soybean genome, while most of the PEPC isoforms are functionally unknown. In this study, we investigated on soybean PEPC expressed in the external layer of seed coat (ELSC) during seed formation. PEPC activity in ELSC ranged from 0.24 to 1.0U/g F.W., which could be comparable to those in whole seeds at U per dry matter. Public RNA-Seq data in separated soybean seed tissues revealed that six plant-type PEPC isogenes were substantially expressed in ELSC, and Gmppc1 and Gmppc7 were highly expressed in hourglass cells of ELSC. Gene Ontology enrichment of co-expressed genes with Gmppc1 and Gmppc7 implicated a role of these isogenes in assisting energy production and cellulose biosynthesis. Comparison of PEPC sequences from 16 leguminous species hypothesized adaptive evolution of the Gmppc1 and Gmppc7 lineage after divergence from the other plant-type PEPC lineages. Molecular diversification of these plant-type PEPC was possibly accomplished by adaptation to the functions of the soybean seed tissues. This study indicates that energy demand in immature seeds may be a driving force for the molecular evolution of PEPC.

The glucose uptake systems in Corynebacterium glutamicum: a review.

The phosphoenolpyruvate-dependent glucose phosphotransferase system (PTSGlc) is the major uptake system responsible for transporting glucose, and is involved in glucose translocation and phosphorylation in Corynebacterium glutamicum. For the longest time, the PTSGlc was considered as the only uptake system for glucose. However, some PTS-independent glucose uptake systems (non-PTSGlc) were discovered in recent years, such as the coupling system of inositol permeases and glucokinases (IPGS) and the coupling system of β-glucoside-PTS permease and glucokinases (GPGS). The products (e.g. lysine, phenylalanine and leucine) will be increased because of the increasing intracellular level of phosphoenolpyruvate (PEP), while some by-products (e.g. lactic acid, alanine and acetic acid) will be reduced when this system become the main uptake pathway for glucose. In this review, we survey the uptake systems for glucose in C. glutamicum and their composition. Furthermore, we summarize the latest research of the regulatory mechanisms among these glucose uptake systems. Detailed strategies to manipulate glucose uptake system are addressed based on this knowledge.