Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Starch synthesis and CO₂ evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with ¹⁴C-labelled hexose-phosphates. Amyloplasts converted [U-¹⁴C]glucose 1-phosphate (Glc1P) but not [U-¹⁴C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-¹⁴C]Glc1P and [U-¹⁴C]Glc6P were metabolized to CO₂, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium. Received: 29 March 1997 / Accepted: 5 June 1997     

Isoenzyme studies in one Brazilian and two Venezuelan strains of Schistosoma mansoni.

1. Enzyme polymorphism, analyzed by starch gel electrophoresis, was found to be zero for acid phosphatase, phosphoglucomutase, phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and malic enzyme, in one Brazilian and two Venezuelan strains of Schistosoma mansoni. 2. All loci studied were monomorphic within strains, but the isoenzymic patterns were, however, different among the strains. 3. Results suggest a drastic loss of the genetic variability usually found in natural populations.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

The sources of carbon and reducing power for fatty acid synthesis in the heterotrophic plastids of developing sunflower (Helianthus annuus L.) embryos

The provision of carbon substrates and reducing power for fatty acid synthesis in the heterotrophic plastids of developing embryos of sunflower (Helianthus annuus L.) has been investigated. Profiles of oil and storage protein accumulation were determined and embryos at 17 and 24 days after anthesis (DAA) were selected to represent early and late periods of oil accumulation. Plastids isolated from either 17 or 24 DAA embryos did not incorporate label from [1-(14)C]glucose 6-phosphate (Glc6P) into fatty acids. Malate, when supplied alone, supported the highest rates of fatty acid synthesis by the isolated plastids at both stages. Pyruvate supported rates of fatty acid synthesis at 17 DAA that were comparable to those supported by malate, but only when incubations also included Glc6P. The stimulatory effect of Glc6P on pyruvate utilization at 17 DAA was related to the rapid utilization of Glc6P through the oxidative pentose phosphate pathway (OPPP) at this stage. Addition of pyruvate to incubations containing [1-(14)C]Glc6P increased OPPP activity (measured as (14)CO(2) release), while the addition of malate suppressed it. Observations of the interactions between the rate of metabolite utilization for fatty acid synthesis and the rate of the OPPP are consistent with regulation of the OPPP by redox control of the plastidial glucose 6-phosphate dehydrogenase activity through the demand for NADPH. During pyruvate utilization for fatty acid synthesis, flux through the OPPP increases as NADPH is consumed, whereas during malate utilization, in which NADPH is produced by NADP-malic enzyme, flux through the OPPP is decreased.     

Enzymes of carbohydrate metabolism ofCotugnia digonopora and their activity in the presence of anthelmintics,in vitro

Cotugnia digonopora, a fowl cycllophyllidean cestode, was found to possess most of the enzymes, associated with the glycolytic sequence and phosphoenolpyruvate branch point, in the cytosol fraction. Enzymes of malate metabolism were predominantly mitrochondrial. Anthelmintic agents inhibited hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, fumarate reductase, and malic enzyme. In intact worms this effect was significantly reduced. However, the activities of glycogen Phosphorylase and pyruvate kinase were significantly enhanced. Communication No. 4113 from CDRI, Lucknow.     

Activities of Enzymes of Carbohydrate and Energy Metabolism of the Spores of the Microsporidian, Nosema grylli

ABSTRACT. The presence of 14 enzymes was investigated using purified spores of the microsporidian Nosema grylli from fat body of the crickets Gryllus bimaculatus . Glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphoglucomutase (EC 5.4.2.2), phosphoglucose isomerase (EC 5.3.1.9), fructose 6-phosphate kinase (EC 2.7.1.11), aldolase (EC 4.1.2.13), 3-phosophoglycerate kinase (EC 2.7.2.3), pyruvate kinase (EC 2.7.1.40) and glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) were detected with activities of 15 ± 1, 7 ± 1, 1,549 ± 255, 10 ± 1, 5 ± 1, 16 ± 4, 6 ± 1 and 16 ± 2 nmol/min. mg protein, respectively. Hexokinase (EC 2.7.1.1), NAD-dependent malate dehydrogenase (EC 1.1.1.37), malic enzyme (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27), alcohol dehydrogenase (EC 1.1.1.1) and succinate dehydrogenase (EC 1.3.99.1) were not detectable. These results suggest the catabolism of carbohydrates in microsporidia occurs via the Embden-Meyerhof pathway. Glycerol 3-phosphate dehydrogenase may reoxidize NADH which is produced by glyceraldehyde 3-phosphate dehydrogenase in glycolysis.     

Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet

Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 microgram/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme.     

Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess

The aim of this work was to investigate the physiology of Kluyveromyces marxianus CBS 6556 in terms of its low tendency to form ethanol under exposure to sugar excess, and the split of carbon flux which takes place at the level of glucose-6-phosphate. Measurements were performed in batch cultivations, and after a glucose or a lactose pulse applied to chemostat-grown respiring cells (with a dilution rate of 0.1 h(-1)). No ethanol formation was observed in batch cultivations or during pulse experiments, unless the oxygen supply was shut down, indicating that this organism is more strictly Crabtree-negative than its close relative K. lactis and other known Crabtree-negative yeasts. During the pulse experiments, activities of phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase in cell-free extracts remained rather constant, at higher levels than those of Saccharomyces cerevisiae grown at similar conditions. When cells were exposed to glucose concentrations as high as 26 gl(-1), the activity of phosphoglucomutase was higher than that in cells exposed to 14 gl(-1) glucose, whereas the activities of phosphoglucoisomerase and glucose-6-phosphate dehydrogenase did not change. Our results suggest that the low tendency for ethanol formation in K. marxianus might be a consequence of this yeast's capacity of keeping the glycolytic flux constant, due at least in part to the diversion of carbon flux towards the biosynthesis of carbohydrates and towards the pentose phosphate pathway.     

“Sink” regulation of photosynthetic metabolism in transgenic tobacco plants expressing yeast invertase in their cell wall involves a decrease of the Calvin-cycle enzymes and an increase of glycolytic enzymes

Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the sink regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO₂. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.Abbreviations Fru 1,6bisP fructose-1,6-bisphosphate - Fru 1,6bisPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc 1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NADP-GAPDH NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - PFK phosphofructokinase - PEP phosphoenolpyruvate - PFP pyrophosphate:fructose-6-phosphate phosphotransferase - PGA glycerate-3-phosphate - PK pyruvate kinase - Pi inorganic phosphate - Ru1,5bisP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SPS sucrose-phosphate synthase - triose-P triose-phosphates     

An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase

Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required.     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Respiration of mitochondria isolated from differentiated and undifferentiated HT29 colon cancer cells in the presence of various substrates and ADP generating systems

1. Oxygen consumption was investigated in two cultured subpopulations of either undifferentiated (Glc+ cells) or differentiated (Glc- cells) HT29 colon cancer cells and in the corresponding isolated mitochondria. In Glc+ cells, a decrease of the respiration is induced by the presence of glucose (Crabtree effect), whereas it is not the case in Glc- cells. 2. The oxidative phosphorylation rate of Glc- mitochondria is found to be much higher than that of Glc+ mitochondria, due to a higher efficiency to oxidize glutamine, glutamate, 2-oxoglutarate, succinate or malate. 3. In both types of mitochondria, respiration can be supported by the ADP formed by adenylate kinase or nucleotide diphosphate kinase, and, although to a lesser extent in Glc- mitochondria, by hexokinase. 4. Glc+ cells are characterized by a low respiration capacity and a high glycolytic flux leading to the Crabtree effect. Glc- cells are characterized by a better correlation between a moderate glycolytic flux and a high respiratory capacity.     

Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos

32P-labeled glucose 6-phosphate and phosphoenolpyruvate were injected into oocytes, fertilized eggs, and early embryos of Xenopus laevis, and the 32P label was followed into glycolytic enzymes and acid-soluble metabolites. The kinetics of labeling of phosphoglucomutase and phosphoglyceromutase and the formation of specific metabolites were used to measure carbon flux through glycolytic intermediates in these cells. In full-grown stage VI oocytes, fertilized eggs, and cells of cleaving embryos, carbon metabolism is in the glycogenic direction. Glycolytic intermediates injected into these cells were metabolized into UDP-glucose and then presumably into glycogen. Carbon flow between phosphoenolpyruvate and glucose 6-phosphate does not utilize fructose 1,6-bisphosphatase; rather, it may depend largely on enzymes of the pentose phosphate pathway. Maturation and fertilization of the oocyte did not result in a change in the qualitative pattern of metabolites formed. Pyruvate kinase, although abundant in oocytes and embryos, is essentially inactive in these cells. Pyruvate kinase also appears to be inactive in small previtellogenic stage II oocytes; however, in these cells injected glycolytic intermediates were not metabolized to UDP-glucose.     

Role and location of NAD malic enzyme in thermogenic tissues of Araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.     

Malate utilization by a group D Streptococcus: physiological properties and purification of an inducible malic enzyme

Growth of Streptococcus faecalis in the presence of l-malate resulted in the induction of a "malic enzyme" [l-malate:nicotinamide adenine dinucleotide (NAD) oxidoreductase (decarboxylating), E.C. 1.1.1.39]. Synthesis of the malic enzyme did not appear to be subject to catabolite repression by intermediate products of glucose or fructose dissimilation. However, malate utilization was inhibited during growth in the presence of glucose or fructose. The purified enzyme was specific for malate as substrate and NAD as cofactor. Mn(+2) or Mg(+2) was required for optimal activity and NH(4)Cl stimulated the reaction rate. Several lines of indirect evidence suggested that the streptococcal malic enzyme was involved primarily with energy production and not biosynthesis.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Analysis of carbon metabolism in Escherichia coli strains with an inactive phosphotransferase system by (13)C labeling and NMR spectroscopy

We have developed Escherichia coli strains that internalize glucose utilizing the GalP permease instead of the phosphoenolpyruvate:carbohydrate phosphotransferase system. It has been demonstrated that a strain with these modifications (PTS(-)Glc(+)) can direct more carbon flux into the aromatic pathway than the wild-type parental strain (N. Flores et al., 1996, Nat. Biotechnol. 14, 620-623; G. Gosset et al., 1996, J. Ind. Microbiol. 17, 47-52; J. L. Baéz et al., 2001, Biotechnol. Bioeng. 73, 530-535). In this study, we have determined and compared the carbon fluxes of a wild-type strain (JM101), a PTS(-)Glc(-) strain, and two isogenic PTS(-)Glc(+) derivatives named PB12 and PB13 by combining genetic, biochemical, and NMR approaches. It was determined that in these strains a functional glk gene in the chromosome is required for rapid glucose consumption; furthermore, glucokinase-specific activities were higher than in the wild-type strain. (13)C labeling and NMR analysis allowed the determination of differences in vivo which include higher glycolytic fluxes of 93.1 and 89.2% compared with the 76.6% obtained for the wild-type E. coli. In PB12 and PB13 we found a flux through the malic enzymes of 4 and 10%, respectively, compared to zero in the wild-type strain. While flux through the Pck enzyme was absent in PB12 and PB13, in the wild type it was 7.7%. Finally, it was found that in the JM101 and PB12 strains both the oxidative and the nonoxidative branches of the pentose phosphate pathway contributed to ribose 5-phosphate synthesis, whereas in PB13 this pentose was synthesized almost exclusively through the oxidative branch. The determined carbon fluxes correlate with biochemical and genetic characterizations.