scholarly journals Novel Listerial Glycerol Dehydrogenase- and Phosphoenolpyruvate-Dependent Dihydroxyacetone Kinase System Connected to the Pentose Phosphate Pathway

2012 ◽  
Vol 194 (18) ◽  
pp. 4972-4982 ◽  
Author(s):  
Céline Monniot ◽  
Arthur Constant Zébré ◽  
Francine Moussan Désirée Aké ◽  
Josef Deutscher ◽  
Eliane Milohanic
Keyword(s):  
Pentose Phosphate Pathway ◽  
Triosephosphate Isomerase ◽  
Pentose Phosphate ◽  
Glycerol Dehydrogenase ◽  
Phosphoryl Group ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Content Type ◽  
New Type ◽  
Enzyme I

ABSTRACTSeveral bacteria use glycerol dehydrogenase to transform glycerol into dihydroxyacetone (Dha). Dha is subsequently converted into Dha phosphate (Dha-P) by an ATP- or phosphoenolpyruvate (PEP)-dependent Dha kinase.Listeria innocuapossesses two potential PEP-dependent Dha kinases. One is encoded by 3 of the 11 genes forming the glycerol (gol) operon. This operon also containsgolD(lin0362), which codes for a new type of Dha-forming NAD+-dependent glycerol dehydrogenase. The subsequent metabolism of Dha requires its phosphorylation via the PEP:sugar phosphotransferase system components enzyme I, HPr, and EIIADha-2 (Lin0369). P∼EIIADha-2 transfers its phosphoryl group to DhaL-2, which phosphorylates Dha bound to DhaK-2. The resulting Dha-P is probably metabolized mainly via the pentose phosphate pathway, because two genes of thegoloperon encode proteins resembling transketolases and transaldolases. In addition, purified Lin0363 and Lin0364 exhibit ribose-5-P isomerase (RipB) and triosephosphate isomerase activities, respectively. The latter enzyme converts part of the Dha-P into glyceraldehyde-3-P, which, together with Dha-P, is metabolized via gluconeogenesis to form fructose-6-P. Together with another glyceraldehyde-3-P molecule, the transketolase transforms fructose-6-P into intermediates of the pentose phosphate pathway. Thegoloperon is preceded bygolR, transcribed in the opposite orientation and encoding a DeoR-type repressor. Its inactivation causes the constitutive but glucose-repressible expression of the entiregoloperon, including the last gene, encoding a pediocin immunity-like (PedB-like) protein. Its elevated level of synthesis in thegolRmutant causes slightly increased immunity against pediocin PA-1 compared to the wild-type strain or apedB-like deletion mutant.

scholarly journals Phosphotransferase System-Mediated Glucose Uptake Is Repressed in Phosphoglucoisomerase-Deficient Corynebacterium glutamicum Strains

2013 ◽  
Vol 79 (8) ◽  
pp. 2588-2595 ◽  
Author(s):  
Steffen N. Lindner ◽  
Dimitar P. Petrov ◽  
Christian T. Hagmann ◽  
Alexander Henrich ◽  
Reinhard Krämer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Glucose Uptake ◽  
Corynebacterium Glutamicum ◽  
Pentose Phosphate ◽  
Phosphotransferase System ◽  
Negative Effects ◽  
Gene Encoding ◽  
Lysine Production ◽  
Content Type ◽  
Model Strain

ABSTRACTCorynebacterium glutamicumis particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of thepgigene, encoding the phosphoglucoisomerase, has been applied for the improvement ofC. glutamicumamino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted thepgigene in the type strainC. glutamicumATCC 13032. The resulting strain,C. glutamicumΔpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake inC. glutamicumΔpgi. Furthermore, Northern blot analyses revealed that expression ofptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimizedl-lysine production in the model strainC. glutamicumDM1729 by deletion ofpgiand overexpression of plasmid-encodedptsG.l-Lysine yields and productivity withC. glutamicumΔpgi(pBB1-ptsG) were significantly higher than those withC. glutamicumΔpgi(pBB1). These results show thatptsGoverexpression is required to overcome the repressed activity of PTS-mediated glucose uptake inpgi-deficientC. glutamicumstrains, thus enabling efficient as well as fastl-lysine production.

scholarly journals Confirmation and Elimination of Xylose Metabolism Bottlenecks in Glucose Phosphoenolpyruvate-Dependent Phosphotransferase System-Deficient Clostridium acetobutylicum for Simultaneous Utilization of Glucose, Xylose, and Arabinose

2011 ◽  
Vol 77 (22) ◽  
pp. 7886-7895 ◽  
Author(s):  
Han Xiao ◽  
Yang Gu ◽  
Yuanyuan Ning ◽  
Yunliu Yang ◽  
Wilfrid J. Mitchell ◽  
...  
Keyword(s):  
Clostridium Acetobutylicum ◽  
Xylose Isomerase ◽  
Cost Effective ◽  
Pentose Phosphate ◽  
Xylose Metabolism ◽  
Phosphotransferase System ◽  
Gene Encoding ◽  
Content Type ◽  
Sugar Mixtures ◽  
Residual Glucose

ABSTRACTEfficient cofermentation ofd-glucose,d-xylose, andl-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacteriumClostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predictedglcGgene, encoding enzyme II of thed-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strainClostridium acetobutylicumATCC 824, resulting in greatly improvedd-xylose andl-arabinose consumption in the presence ofd-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermentedd-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result ofglcGdisruption. Furthermore, the inherent rate-limiting steps of thed-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of thed-xylose proton-symporter (cac1345),d-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integratingglcGdisruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures ofd-glucose,d-xylose, andl-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.

scholarly journals A Single Point Mutation Controls the Rate of Interconversion Between the g+ and g− Rotamers of the Histidine 189 χ2 Angle That Activates Bacterial Enzyme I for Catalysis

2021 ◽  
Vol 8 ◽  
Author(s):  
Jeffrey A. Purslow ◽  
Jolene N. Thimmesch ◽  
Valeria Sivo ◽  
Trang T. Nguyen ◽  
Balabhadra Khatiwada ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Single Point ◽  
Conformational Dynamics ◽  
Phosphorylation Site ◽  
Phosphoryl Group ◽  
Single Point Mutation ◽  
Phosphotransferase System ◽  
Function Relationship ◽  
Enzyme I

Enzyme I (EI) of the bacterial phosphotransferase system (PTS) is a master regulator of bacterial metabolism and a promising target for development of a new class of broad-spectrum antibiotics. The catalytic activity of EI is mediated by several intradomain, interdomain, and intersubunit conformational equilibria. Therefore, in addition to its relevance as a drug target, EI is also a good model for investigating the dynamics/function relationship in multidomain, oligomeric proteins. Here, we use solution NMR and protein design to investigate how the conformational dynamics occurring within the N-terminal domain (EIN) affect the activity of EI. We show that the rotameric g+-to-g− transition of the active site residue His189 χ2 angle is decoupled from the state A-to-state B transition that describes a ∼90° rigid-body rearrangement of the EIN subdomains upon transition of the full-length enzyme to its catalytically competent closed form. In addition, we engineered EIN constructs with modulated conformational dynamics by hybridizing EIN from mesophilic and thermophilic species, and used these chimeras to assess the effect of increased or decreased active site flexibility on the enzymatic activity of EI. Our results indicate that the rate of the autophosphorylation reaction catalyzed by EI is independent from the kinetics of the g+-to-g− rotameric transition that exposes the phosphorylation site on EIN to the incoming phosphoryl group. In addition, our work provides an example of how engineering of hybrid mesophilic/thermophilic chimeras can assist investigations of the dynamics/function relationship in proteins, therefore opening new possibilities in biophysics.

scholarly journals Role of Intracellular Carbon Metabolism Pathways in Shigella flexneri Virulence

2014 ◽  
Vol 82 (7) ◽  
pp. 2746-2755 ◽  
Author(s):  
E. A. Waligora ◽  
C. R. Fisher ◽  
N. J. Hanovice ◽  
A. Rodou ◽  
E. E. Wyckoff ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Plaque Assay ◽  
Pentose Phosphate ◽  
Host Cells ◽  
Content Type ◽  
Pathway Gene

ABSTRACTShigella flexneri, which replicates in the cytoplasm of intestinal epithelial cells, can use the Embden-Meyerhof-Parnas, Entner-Doudoroff, or pentose phosphate pathway for glycolytic carbon metabolism. To determine which of these pathways is used by intracellularS. flexneri, mutants were constructed and tested in a plaque assay for the ability to invade, replicate intracellularly, and spread to adjacent epithelial cells. Mutants blocked in the Embden-Meyerhof-Parnas pathway (pfkABandpykAFmutants) invaded the cells but formed very small plaques. Loss of the Entner-Doudoroff pathway geneedaresulted in small plaques, but the doubleeda eddmutant formed normal-size plaques. This suggested that the plaque defect of theedamutant was due to buildup of the toxic intermediate 2-keto-3-deoxy-6-phosphogluconic acid rather than a specific requirement for this pathway. Loss of the pentose phosphate pathway had no effect on plaque formation, indicating that it is not critical for intracellularS. flexneri. Supplementation of the epithelial cell culture medium with pyruvate allowed the glycolysis mutants to form larger plaques than those observed with unsupplemented medium, consistent with data from phenotypic microarrays (Biolog) indicating that pyruvate metabolism was not disrupted in these mutants. Interestingly, the wild-typeS. flexnerialso formed larger plaques in the presence of supplemental pyruvate or glucose, with pyruvate yielding the largest plaques. Analysis of the metabolites in the cultured cells showed increased intracellular levels of the added compound. Pyruvate increased the growth rate ofS. flexneriin vitro, suggesting that it may be a preferred carbon source inside host cells.

scholarly journals Combined Fluxomics and Transcriptomics Analysis of Glucose Catabolism via a Partially Cyclic Pentose Phosphate Pathway in Gluconobacter oxydans 621H

2013 ◽  
Vol 79 (7) ◽  
pp. 2336-2348 ◽  
Author(s):  
Tanja Hanke ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
Tino Polen ◽  
Stephanie Bringer ◽  
...  
Keyword(s):  
Phase Ii ◽  
Growth Phase ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Glucose Dehydrogenase ◽  
Gluconobacter Oxydans ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Content Type ◽  
Activity Measurements

ABSTRACTIn this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes inGluconobacter oxydans621H with glucose were studied by13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For13C-MFA, cells were cultivated with specifically13C-labeled glucose, and intracellular metabolites were analyzed for their labeling pattern by liquid chromatography-mass spectrometry (LC-MS). In growth phase I, 90% of the glucose was oxidized periplasmically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. SinceG. oxydanslacks phosphofructokinase, glucose 6-phosphate can be metabolized only via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP).13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phases I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.

scholarly journals Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Max Chavarría ◽  
Roelco J. Kleijn ◽  
Uwe Sauer ◽  
Katharina Pflüger-Grau ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Metabolic Networks ◽  
Tca Cycle ◽  
High Energy ◽  
Pentose Phosphate ◽  
Cell Physiology ◽  
Phosphotransferase System ◽  
Content Type ◽  
Metabolic Fluxes ◽  
Central Carbon

ABSTRACTTwo branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacteriumPseudomonas putidaKT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFruor PTSNtrgenes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtrprotein of the PTSNtrthus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology.IMPORTANCEThis study demonstrates that EIIANtrinfluences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars byP. putida. These findings indicate that the EIIANtrprotein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

scholarly journals The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Laure Dumont ◽  
Mark B. Richardson ◽  
Phillip van der Peet ◽  
Danushka S. Marapana ◽  
Tony Triglia ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
Pentose Phosphate ◽  
Repair Enzyme ◽  
Content Type ◽  
Phosphoglycolate Phosphatase ◽  
Central Carbon ◽  
Metabolite Repair ◽  
By Products

ABSTRACT Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparum pgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite. IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.

scholarly journals Distribution and Functions of Phosphotransferase System Genes in the Genome of the Lactic Acid Bacterium Oenococcus oeni

2013 ◽  
Vol 79 (11) ◽  
pp. 3371-3379 ◽  
Author(s):  
Zohra Jamal ◽  
Cécile Miot-Sertier ◽  
François Thibau ◽  
Lucie Dutilh ◽  
Aline Lonvaud-Funel ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Oenococcus Oeni ◽  
Phosphotransferase System ◽  
Content Type ◽  
Low Concentrations ◽  
Perfect Adaptation ◽  
Form Part ◽  
Genes Encoding ◽  
Enzyme I

ABSTRACTOenococcus oeni, the lactic acid bacterium primarily responsible for malolactic fermentation in wine, is able to grow on a large variety of carbohydrates, but the pathways by which substrates are transported and phosphorylated in this species have been poorly studied. We show that the genes encoding the general phosphotransferase proteins, enzyme I (EI) and histidine protein (HPr), as well as 21 permease genes (3 isolated ones and 18 clustered into 6 distinct loci), are highly conserved among the strains studied and may form part of theO. oenicore genome. Additional permease genes differentiate the strains and may have been acquired or lost by horizontal gene transfer events. The coreptsgenes are expressed, and permease gene expression is modulated by the nature of the bacterial growth substrate. DecryptifiedO. oenicells are able to phosphorylate glucose, cellobiose, trehalose, and mannose at the expense of phosphoenolpyruvate. These substrates are present at low concentrations in wine at the end of alcoholic fermentation. The phosphotransferase system (PTS) may contribute to the perfect adaptation ofO. oenito its singular ecological niche.

scholarly journals Mannitol and the Mannitol-Specific Enzyme IIB Subunit Activate Vibrio cholerae Biofilm Formation

2013 ◽  
Vol 79 (15) ◽  
pp. 4675-4683 ◽  
Author(s):  
Patrick Ymele-Leki ◽  
Laetitia Houot ◽  
Paula I. Watnick
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Diarrheal Disease ◽  
Ectopic Expression ◽  
Phosphotransferase System ◽  
Content Type ◽  
Regulatory Pathways ◽  
B Subunit ◽  
Multicomponent Signal ◽  
Enzyme I

ABSTRACTVibrio choleraeis a halophilic, Gram-negative rod found in marine environments. Strains that produce cholera toxin cause the diarrheal disease cholera.V. choleraeuse a highly conserved, multicomponent signal transduction cascade known as the phosphoenolpyruvate phosphotransferase system (PTS) to regulate carbohydrate uptake and biofilm formation. Regulation of biofilm formation by the PTS is complex, involving many different regulatory pathways that incorporate distinct PTS components. The PTS consists of the general components enzyme I (EI) and histidine protein (HPr) and carbohydrate-specific enzymes II. Mannitol transport byV. choleraerequires the mannitol-specific EII (EIIMtl), which is expressed only in the presence of mannitol. Here we show that mannitol activatesV. choleraebiofilm formation and transcription of thevpsbiofilm matrix exopolysaccharide synthesis genes. This regulation is dependent on mannitol transport. However, we show that, in the absence of mannitol, ectopic expression of the B subunit of EIIMtlis sufficient to activate biofilm accumulation. Mannitol, a common compatible solute and osmoprotectant of marine organisms, is a main photosynthetic product of many algae and is secreted by algal mats. We propose that the ability ofV. choleraeto respond to environmental mannitol by forming a biofilm may play an important role in habitat selection.

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