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.     

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.     

Preferential use of glucose during diauxic growth of Carrot cells growing on glucose and malate

Suspension cells of carrot plants grown on mixed carbon sources of glucose (Glc) and malate preferentially used Glc. The cells started to utilize malate only after Glc was depleted from the medium, thus exhibiting a diauxic growth. The residual concentration of Glc decreased rapidly during the first growth phase, and that of malate decreased only during the second growth phase. Malate uptake was negligible throughout the diauxic growth, suggesting that malate was being utilized via another metabolite. An active metabolic flow from fumarate to pyruvate and oxaloacetate via malate was induced in cells during the second growth phase. These results strongly suggested that malate remained unused in the medium in the first phase, and in the second phase it was converted extracellularly into fumarate, which was subsequently transported into cells and metabolized into malate and further into pyruvate and oxaloacetate. This study presents the second case of diauxic growth in plants and the peculiar mode of malate utilization.     

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.     

Production of L-malate from fumarate by the yeast Dipodascus magnusii

An alternative microbiological method for the production of malate from fumarate is presented. The yeast Dipodascus magnusii was used for this bioconversion. The optimum cell growth temperature was 28°C and the working volume 120 ml. The highest level of fumarase activity during bioconversion was achieved at a pH of 7.5 and a temperature of 37°C. These conditions were determined as optimal. Using sodium fumarate (1M), the maximum specific productivity of malic acid obtained was 1.72 g/(g_DCW × h) for intact cells. In the case of ammonium fumarate, it was 2.25 g/(g_DCW × h).     

Cloning, sequencing, and mutational analysis of the Bradyrhizobium japonicum fumC-like gene: evidence for the existence of two different fumarases.

The Bradyrhizobium japonicum fumarase gene (fumC-like) was cloned and sequenced, and a fumC deletion mutant was constructed. This mutant had a Nod+ Fix+ phenotype in symbiosis with the host plant, soybean, and growth in minimal medium with fumarate as sole carbon source was also not affected. The cloned B. japonicum fumC gene fully complemented an Escherichia coli Fum- mutant, strain JH400, for growth in minimal medium with fumarate. The predicted amino acid sequence of the FumC protein showed strong similarity to the E. coli FumC protein, Bacillus subtilis CitG protein, Saccharomyces cerevisiae Fum1 protein, and the mammalian fumarases. The B. japonicum FumC protein accounted for about 40% of the total fumarase activity in aerobically grown cells. The remaining 60% was ascribed to a temperature-labile fumarase. These data suggest that B. japonicum possesses two different fumarase isoenzymes, one of which is encoded by fumC. Besides E. coli, which has three fumarases, B. japonicum is thus the second bacterium for which there is genetic evidence for the existence of more than one fumarase.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Acid excreting mutants of yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants acidifying glucose medium containing bromocresol purple were shown to excrete protons when placed in unbuffered water in the absence of any external carbon source. The mutants belong to 16 different complementation groups. Most of them do not grow on glycerol and the excreted protons are associated to particular sets of organic anions such as citrate, aconitate, succinate, fumarate or malate. These novel types of respiratory mutations seem to be located in genes operating in the Krebs or glyoxylate cycle.     

L-malate production by metabolically engineered Escherichia coli

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts.     

Succinate uptake and related proton movements in Escherichia coli K12.

1. The apparent Km values for succinate uptake by whole cells of Escherichia coli K12 depend on pH in the range 6.5-7.4.2. Uptake of succinate in lightly buffered medium is accompanied by proton uptake. 3. The apparent Km values for succinate uptake and for succinate-induced proton uptake are similar. 4. Approximately two protons enter the cell with each succinate molecule. 5. The pattern of inhibition of succinate uptake is similar to that of succinate-induced proton uptake. 6. Uptake of fumarate and malate, which share the succinate-transport system, is also accompanied by the uptake of approximately two protons per molecule of fumarate or malate. 7. Uptake of aspartate by the dicarboxylic acid-transport system is accompanied by the uptake of approximatley two protons per molecule of asparatate. 8. It is concluded that uptake of dicarboxylic acids by the dicarboxylic acid-transport system is obligatorily coupled to proton uptake such that succinate, malate and fumarate are taken up in electroneutral form and asparate is taken up in cationic form. 9. These results are consistent with, though they do not definitely prove, the energization of succinate uptake of the deltapH.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Chicken fumarase. II. Kinetic studies.

The catalysis of the hydration of fumarate and deshydration of L - malate by chicken fumarase was measured spectrophotometrically over a range of substrate concentrations from 4 times 10(-3) M to 8 times 10(-5) M for fumarate and from 8 times 10(-2) M to 10(-3) M for L - malate. For the forward and reverse reactions, linear Lineweaver and Burk plots were obtained. The Michaelis constants and the maximum initial velocities for both substrates were determined and the Haldane relation was found to be obeyed. The effect of pH on activity was investigated over a pH range from 5.5 to 9.0 and the data indicate the presence, in the active site, of two ionizable groups, one in the acidic form and one in the basic form. The values of the ionization constants, determined for the enzyme - substrate complexes, agree closely with the ones obtained for the porcine enzyme. The mode of action of twenty-four structural analogs on the initial velocity of the dehydration of L-malate, by chicken fumarase was examined. From these studies, two regions positively charged appear necessary for the effective binding of the carboxylates of the substrates and competitive inhibitors to the active center. Moreover, the data suggest the presence of an additional group, in the catalytic site of chicken fumarase, that stabilizes the carbon-carbon double bond common to fumarate and its structural analogs. Finally, from the comparison of the kinetic properties of the chicken and pig fumarases, it may be concluded that the catalytic mechanism of the homologous enzymes are very similar, if not identical.     

Equilibration of Label in Malate during Dark Fixation of CO(2) in Kalanchoë fedtschenkoi

In vitro studies of dark ¹⁴CO₂ fixation with isolated cell aggregates of Kalanchoë fedtschenkoi showed that malate synthesized after 20 sec is predominantly (85 to 92%) labeled at carbon 4, while after 20 min only 65 to 69% of the radioactivity was located in this position. The intramolecular labeling pattern of malate could not be changed by supplementing the cells with carboxylation reaction substrates such as ribulose diphosphate or phosphoenolpyruvate. The kinetic decline of label at carbon 4 of malate occurs independently of CO₂ fixation, since 4-¹⁴C-labeled aspartate fed to the cells gave rise to malate labeled 62% at carbon 4 after 20 min. Furthermore, the cells were capable of converting fed malate to fumarate. It is concluded that synthesis of malate during dark CO₂ fixation is accomplished by a single carboxylation step via phosphoenolpyruvate carboxylase and labeling patterns observed in malate are a consequence of the action of fumarase.     

Identification of the catalytic mechanism and estimation of kinetic parameters for fumarase

The enzyme fumarase catalyzes the reversible hydration of fumarate to malate. The reaction catalyzed by fumarase is critical for cellular energetics as a part of the tricarboxylic acid cycle, which produces reducing equivalents to drive oxidative ATP synthesis. A catalytic mechanism for the fumarase reaction that can account for the kinetic behavior of the enzyme observed in both isotope exchange studies and initial velocity studies has not yet been identified. In the present study, we develop an 11-state kinetic model of the enzyme based on the current consensus on its catalytic mechanism and design a series of experiments to estimate the model parameters and identify the major flux routes through the mechanism. The 11-state mechanism accounts for competitive binding of inhibitors and activation by different anions, including phosphate and fumarate. The model is identified from experimental time courses of the hydration of fumarate to malate obtained over a wide range of buffer and substrate concentrations. Further, the 11-state model is found to effectively reduce to a five-state model by lumping certain successive steps together to yield a mathematically less complex representation that is able to match the data. Analysis suggests the primary reaction route of the catalytic mechanism, with fumarate binding to the free unprotonated enzyme and a proton addition prior to malate release in the fumarate hydration reaction. In the reverse direction (malate dehydration), malate binds the protonated form of the enzyme, and a proton is generated before fumarate is released from the active site.     

A naturally occurring promoter polymorphism of the Arabidopsis FUM2 gene causes expression variation,and is associated with metabolic and growth traits

Fumarate and malate are known intermediates of the TCA cycle, a mitochondrial metabolic pathway generating NADH for respiration. Arabidopsis thaliana and other Brassicaceae contain an additional cytosolic fumarase (FUM2) that functions in carbon assimilation and nitrogen use. Here, we report the identification of a hitherto unknown FUM2 promoter insertion/deletion (InDel) polymorphism found between the Col‐0 and C24 accessions, which also divides a large number of Arabidopsis accessions carrying either the Col‐0 or the C24 allele. The polymorphism consists of two stretches of 2.1 and 3.8 kb, which are both absent from the promotor region of Col‐0 FUM2. By analysing mutants as well as mapping and natural populations with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate/malate ratio in leaves. In a large population of 174 natural accessions, the polymorphism was also found to be associated with the fumarate/malate ratio, malate and fumarate levels, and with dry weight at 15 days after sowing (DAS). The association with biomass production was confirmed in an even larger (251) accession population for dry weight at 22 DAS. The dominant Col‐0 allele that results in increased fumarate/malate ratios and enhanced biomass production is predominantly found in central/eastern European accessions, whereas the C24 type allele is prevalent on the Iberian Peninsula, west of the Rhine and in the British Isles. Our findings support the role of FUM2 in diurnal carbon storage, and point to a growth advantage of accessions carrying the FUM2 Col‐0 allele.     

Expression and properties of the mitochondrial and cytosolic forms of fumarase in germinating maize seeds

Fumarase (EC 4.2.1.2) catalyzes reversible interconversion of malate and fumarate. It is usually associated with the tricarboxylic acid cycle in mitochondria, although the cytosolic form has also been detected. We investigated the expression of two fumarase genes and activities of the mitochondrial and cytosolic isoforms of fumarase in maize (Zea mays) scutellum during germination. Both isoforms were purified to electrophoretic homogeneity. The cytosolic form had low optimum pH (6.5) and high affinity to malate (K_m 5 μM) when compared with the mitochondrial form (optimum pH 7.0, K_m 50 μM). The cytosolic form was strongly activated by Mg²⁺ and even more by Mn²⁺, whereas the mitochondrial form was moderately activated by Mg²⁺ and Mn²⁺ was less effective. The highest fumarase activity in scutellum and a high expression of the gene encoding the cytosolic form were observed during the maximal activity of the glyoxylate cycle. In leaves, the localization of fumarase is only mitochondrial and only one fumarase gene is expressed. It is concluded that the function of cytosolic fumarase in maize scutellum can be related to metabolism of succinate formed in the glyoxylate cycle.     

Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from ¹⁴C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.     

Regulation of the dicarboxylic acid part of the citric acid cycle in Bacillus subtilis.

The regulation of alpha-ketogluterate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase, and malic enzyme has been studied in Bacillus subitilis. The levels of these enzymes increase rapidly during late exponential phase in a complex medium and are maximal 1 to 2 h after the onset of sporulation. Regulation of enzyme synthesis has been studied in the wild type and different citric acid cycle mutants by adding various metabolites to the growth medium. Alpha-ketoglutarate dehydrogenase is induced by glutamate or alpha-ketoglutarate; succinate dehydrogenase is repressed by malate; and fumarase and malic enzyme are induced by fumarate and malate, respectively. The addition of glucose leads to repression of the citric acid cycle enzymes whereas the level of malic enzyme is unaffected. Studies on the control of enzyme activities in vitro have shown that alpha-ketoglutarate dehydrogenase and succinate dehydrogenase are inhibited by oxalacetate. Enzyme activities are also influenced by the energy level, expressed as the energy charge of the adenylate pool. Isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malic enzyme are inhibited at high energy charge values, whereas malate dehydrogenase is inhibited at low energy charge. A survey of the regulation of the citric acid cycle in B.subtilis, based on the present work and previously reported results, is presented and discussed.     

Studies on xanthan production from Xanthomonas campestris

Xanthan is an heteropolysaccharide produced by Xanthomonascampestris. Xanthan gum fermentation by a local isolate of X. campestris using different carbon sources was studied. The production of polysaccharide was influenced by the carbon source used. The production of the xanthan was 15.654 g/l with synthetic medium. Production of xanthan at various temperatures ranging between 25v°C and 40v°C was studied. The growth and production was maximum between 25-30v°C. Xanthan production was maximum at pH 7.0-7.5.