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PubMed2003

Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations.

Metabolic flux analysis based on 13C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF- mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF- mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.

Acetic Acid↗
PubMed2003

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities.

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner-Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on 13C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.

Carbohydrate Metabolism↗
PubMed2006

Production and partial characterization of arabinoxylan-degrading enzymes by Penicillium brasilianum under solid-state fermentation.

The production of a battery of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum grown on brewer's spent grain (BSG) under solid-state fermentation was investigated. Initial moisture content, initial pH, temperature, and nitrogen source content were optimized to achieve maximum production of feruloyl esterase, xylanase, and alpha-L: -arabinofuranosidase. Under the optimum growth conditions (80% moisture, pH 6, 26.5 degrees C, and 5 g/l nitrogen source), the maximum level of feruloyl esterase (1,542 mU/g BSG) was found after 196 h, whereas xylanase (709 U/g BSG) and ArabF activity (3,567 mU/g BSG) were maximal after 108 h and 96 h, respectively. Based on substrate utilization data, the feruloyl esterases produced by P. brasilianum was anticipated to subclass B. A crude enzyme (CE) preparation from P. brasilianum culture grown on BSG was tested for the release of hydroxycinnamic acids and pentoses from BSG. The P. brasilianum CE produced in this work contains a balance of cell wall-modifying enzymes capable of degrading arabinoxylan of BSG by more than 40%.

Carboxylic Ester Hydrolases↗
PubMed2006

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae.

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.

Aldehyde Reductase↗
PubMed2000

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum.

A recently developed method for analyzing metabolic networks using 13C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69+/-3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA.

Acetates↗
PubMed2005

Comparative chemical characterization of pigmented and less pigmented cell walls of Alternaria tenuissima.

Alternaria tenuissima, the parasitic fungus, was obtained from the pruned upper-cut surfaces of mulberry stems. This fungus contains dark pigment because of the presence of melanin in the cell wall. To obtain less-pigmented cell walls, this fungus was grown under dark condition. When the pigmented and less-pigmented cell walls were chemically analyzed, no differences were observed in amino-acid composition, hexoses, or pentoses. However, in pigmented cell walls, higher contents of melanin (2.6%) were found than in less-pigmented cell walls (0.3%). Interestingly, a significant difference was observed in the relative fatty-acid compositions between these two types of cell walls. Among the major fatty acids, there were increased concentrations of tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), 9-hexadecenoic acid (C16: 1,Delta 9), and 9-octadecanoic acid (C18:1,Delta 9) and a concomitant decrease in 9,12-octadecadienoic acid (C18:2,Delta 9,12) in less-pigmented compared with pigmented cell walls. This difference in fatty-acid composition may be related to the higher percentage of melanin in the pigmented than the less-pigmented cell walls. Lesser amounts of 9,12-octadecadienoic acid in less-pigmented cell walls may have been caused by the growth of the fungus under environmental stress conditions. An interesting observation was the presence in pigmented cell walls only of methyl-substituted fatty acids with carbon numbers C14 to C17, but their occurrence could not be ascertained in the present study.

Alternaria↗
PubMed2001

Poly-beta-hydroxybutyrate metabolism is affected by changes in respiratory enzymatic activities due to cold stress in two psychrotrophic strains of Rhizobium.

Cold stress resulted in a decrease in the poly-beta-hydroxybutyrate (PHB) content of non-cold-acclimated Rhizobium DDSS69 cultures. Analysis of the specific activity of beta-ketothiolase and beta-hydroxybutyrate dehydrogenase revealed that decrease in PHB levels was a result of the inhibition of synthesis of PHB rather than an increase in its breakdown. Rhizobium ATR1, a cold-acclimated strain, revealed the presence of a stable PHB metabolism that did not show any significant differences either in PHB levels or in the activity of enzymes of the PHB metabolism under cold stress, suggesting that PHB is not involved in cold tolerance. Analysis of specific activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway showed the upward regulation of alternate pathways of carbohydrate metabolism under cold stress to rapidly generate energy to overcome the stress. There is diversity in the switching mechanisms of carbon metabolism among cold-acclimated and non-cold-acclimated Rhizobium isolates. Upward regulation of malate dehydrogenase in both isolates suggests that it is a critical input for cold tolerance.

Carbohydrate Metabolism↗
PubMed2004

Grr1p is required for transcriptional induction of amino acid permease genes and proper transcriptional regulation of genes in carbon metabolism of Saccharomyces cerevisiae.

The F-box protein Grr1p is involved in cell cycle regulation, glucose repression and transcriptional induction of the amino acid permease (AAP) gene AGP1. We investigated the role of Grr1p in amino acid-mediated induction of AAP genes by performing batch cultivations with a wild-type strain and a grr1Delta strain and adding citrulline in the exponential phase. Whole-genome transcription analyses were performed on samples from each cultivation, both immediately before and 30 min after citrulline addition. Transcriptional induction of the AAP genes AGP1, BAP2, BAP3, DIP5, GNP1 and TAT1 is fully dependent on Grr1p. Comparison of the grr1Delta strain with the reference strain in the absence of citrulline revealed that GRR1 disruption leads to increased transcription of numerous genes. These encode enzymes in the tricarboxylic acid cycle, the pentose-phosphate pathway and both glucose and starch metabolism. Promoter analysis showed that many of the genes with increased transcription display Mig1p- and/or Msn2p/Msn4p-binding sites. Increased expression of glucose-repressed genes in the grr1Delta strain may be explained by the reduced expression of the hexose transporter genes HXT1, HXT2, HXT3 and HXT4 and a subsequent lowering of the glucose uptake; and the effect of GRR1 deletion on general carbon metabolism may therefore be indirect. Finally, none of the genes known to be primarily involved in cell cycle regulation displayed different expression levels in the grr1Delta cells as compared with the reference strain, suggesting that the role of Grr1p in cell cycle regulation does not include any transcriptional component.

Amino Acid Transport Systems↗
PubMed2006

Comparative study on central metabolic fluxes of Bacillus megaterium strains in continuous culture using 13C labelled substrates.

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by (13)C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.

Bacillus megaterium↗
PubMed1991

Methyl viologen as a preferred electron acceptor in metabolic experiments.

Widely used artificial electron acceptors, including dichlorophenol-indophenol, methylene blue, and phenazine ethosulfate, were shown to induce the nonenzymatic decarboxylation of pyruvate. This characteristic rendered these electron acceptors unsuitable for use in metabolic experiments. In contrast, methyl viologen stimulated the pentose phosphate pathway in mouse preimplantation embryos without decarboxylating pyruvate in the process. Furthermore, methyl viologen had no effect on the Embden-Myerhoff pathway and was not overtly toxic to mouse embryos. These features made methyl viologen a preferred artificial electron acceptor for metabolic studies.

Animals↗
PubMed1992

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.

Amitrole↗
PubMed1992

The role of thiol oxidation in Cobalt(II)-induced toxicity in hamster lung.

Exposure of lung tissue to Co(II) ions both in vivo and in vitro results in toxicity, a relatively early event of which is the oxidation of cellular glutathione. In this study we have attempted to delineate the relationship between this oxidation of glutathione and the subsequent development of cellular dysfunction. Simultaneous incubation with H2O2 potentiated Co(II)-induced increases in both levels of oxidized glutathione (GSSG) and the activity of the pentose phosphate pathway in hamster lung slices. This effect was initially synergistic and, thereafter, both parameters were maintained at significantly greater levels than with either treatment alone throughout the incubation period until the onset of detectable cellular dysfunction. When dysfunction occurred, however, it was not quantitatively increased by the co-treatment over that occurring with CoCl2 alone. Similarly, pretreatment of slices with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) potentiated the Co(II)-induced increase in levels of GSSG. However, this effect was again not associated with an enhancement of cell dysfunction. Since the Co(II)-induced cell damage appeared not to be related directly to the oxidation of glutathione, the quantitative significance of the latter was investigated by comparison with the known oxidant tert-butyl hydroperoxide (t-BOOH). At a concentration of 100 microM, t-BOOH caused an increase in the concentration of GSSG in BCNU-pretreated lung slices which was comparable to that after treatment with Co(II)/H2O2 or CO(II)/BCNU. None of these treatments resulted in a loss of protein thiols. Furthermore, in contrast to Co(II), t-BOOH/BCNU treatment did not result in impaired cell functions. However, at a t-BOOH concentration of 250 microM, t-BOOH/BCNU treatment caused a significantly greater increase in the level of GSSG than that caused by the previous treatments and was associated with both a loss of protein thiols and increased cell dysfunction. We have concluded from these data that under our experimental conditions, Co(II)-induced cell dysfunction is not a consequence of oxidation of cellular glutathione. The reason for this appears to be that the extent of glutathione oxidation by Co(II) even at a concentration which induces cell dysfunction is not of sufficient magnitude to result in the oxidation of protein thiol groups, an event which is likely to constitute the critical consequence of glutathione oxidation in the toxic process.

Animals↗
PubMed1987

DL-apiose substituted with stable isotopes: synthesis, n.m.r.-spectral analysis, and furanose anomerization.

The branched-chain pentose DL-apiose has been synthesized in good yield by a new and simple chemical method that can be adapted to prepare (1-13C)-, (2-13C)-, (1-2H)- and/or (2-2H)-enriched derivatives. N.m.r. spectra (1H- and 13C-) have been interpreted with the aid of selective (13C)- and (2H)-enrichment, and 2D and 13C[13C]-n.m.r. spectra. The solution composition of DL-(1-13C)apiose in 2H2O, determined by 13C-n.m.r. spectroscopy, has been found to differ from that determined previously by 1H-n.m.r. spectroscopy. Several 13C-1H and 13C-13C couplings have been measured and interpreted in terms of apiofuranose ring conformation. Ring-opening rate-constants of the four apiofuranoses [3-C-(hydroxymethyl)-alpha- and -beta-D-erythrofuranose, and 3-C-(hydroxymethyl)-alpha- and -beta-L-threofuranose] have been determined by 13C-saturation-transfer n.m.r. spectroscopy, and compared to those obtained previously for the structurally related tetrofuranoses.

Carbohydrate Conformation↗
PubMed1987

Synthesis and n.m.r.-spectral analysis of unenriched and [1-13C]-enriched 5-deoxypentoses and 5-O-methylpentoses.

Chemical methods are described for preparing unenriched and [1-13C]-enriched 5-deoxy- and 5-O-methyl-pentoses in the D or L configuration. The 1H-n.m.r. spectra of these compounds have been interpreted, and the 13C-n.m.r. spectra assigned with the aid of 2-D 13C-1H chemical-shift correlation spectroscopy. Tautomeric forms (furanoses, hydrate, and aldehyde) in solution in 2H2O have been quantified with the aid of [1-13C]-enriched derivatives. Spectra of 5-deoxypentoses, 5-O-methylpentoses, and methyl pentofuranosides have been compared, in order to assess the effect of 5-C-deoxygenation and 5-O-methylation on chemical shifts and coupling constants (1H-1H, 13C-1H, and 13C-13C) and on the pentofuranose conformations.

Carbohydrate Conformation↗
PubMed1993

Wittig reaction of unprotected D-aldopentoses with stabilized ylides: metal-ion effects.

Each of the four D-aldopentoses (1-4) reacts in the unprotected form with Ph3PCHCO2Me (5) in THF to give stereoselectively the corresponding trans-alpha,beta-unsaturated C7 Wittig adducts, isolated as the corresponding 4,5,6,7-tetraacetates (7-10) or 4,5:6,7-di-O-isopropylidene derivatives (11-14). Concurrently formed are also bicyclic, 1,4-lactone derivatives, isolated as their 5,7-diacetates (15-18) or 5,7-O-isopropylidene derivatives (19), arising through intramolecular Michael addition from the initial acyclic adducts. Formation of the cyclized products is suppressed by incorporation of Cu(OAc)2 in the reaction mixture, permitting preparative isolation of the hept-3-enonate derivatives 7-10, useful as dienophiles in Diels-Alder carbocyclizations with chirality transfer. Optimized yields were 25% (D-ribo), 50% (D-arabino), 49% (D-xylo), and 61% (D-lyxo). Under Cu(OAc)2-catalyzed conditions, the formation of small proportions of 3,7-anhydroheptonic acid esters (21 and 23) was observed in the D-arabino and D-xylo series, but not with the other two pentoses.

Carbohydrate Conformation↗
PubMed1994

In vitro reducing abilities towards chromate of various hydroxy-containing compounds, including saccharides and their derivatives.

The reduction of potassium chromate has been carried out with a variety of OH-containing compounds as reductants, which include pentoses, polyols, glycols, and sugar derivatives. The corresponding reactions were followed using UV-vis and EPR spectroscopies and electrochemistry. The progress of the chromate reduction reactions has been monitored by measuring UV-vis and EPR spectra as a function of time. The observed pseudo first-order reaction rate constants are derived based on the changes in the intensities of the Cr(VI), Cr(V), and Cr(III) signals. Cyclic voltammograms of the simple reductants and their final Cr(III) products formed from the reactions of chromate have also been measured. The reductive abilities of all these reductants have been derived from the spectral data and are discussed on a comparative basis. Based on the results, the aspects that makes a particular reductant more efficient has been addressed. The results obtained from UV-vis, EPR, and cyclic voltammetry are found to be mutually dependent and exhibit among themselves a linear correlation, suggesting that both the reducing and complexing nature of these molecules play important roles in the chromate reduction.

Carbohydrate Conformation↗
PubMed1985

Lack of effect of increased 2,3-diphosphoglycerate on flux through the oxidative pathway in phosphoglycerate kinase deficiency.

Erythrocyte enzyme and substrate levels in two subjects with phosphoglycerate kinase deficiency are reported. The effect of the increased 2,3-diphosphoglycerate, which has been reported as an inhibitor of 6-phosphogluconate dehydrogenase, on the flux through the pentose phosphate pathway, was assessed. There was no significant difference in the flux through erythrocytes from one of the affected subjects and normal subjects in the presence and absence of methylene blue.

2,3-Diphosphoglycerate↗
PubMed1996

Qualitative analysis of biochemical reaction systems.

The qualitative analysis of biochemical reaction systems is presented. A discrete event systems approach is used to represent and analyze bioreaction pathways. The approach is based on Petri nets, which are particularly suited to modeling stoichiometric transformations, i.e. the inter-conversion of metabolites in fixed proportions. The properties and methods for the analysis of Petri nets, along with their interpretation for biochemical systems, are presented. As an example, the combined glycolytic and pentose phosphate pathway of the erythrocyte cell is presented to illustrate the concepts of the methodology.

Animals↗