Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

Effect of aeration on the activity of gluconeogenetic enzymes in Saccharomyces cerevisiae growing under glucose limitation

1. The effect of aeration on the key enzymes of gluconeogenesis was studied in baker's yeast (Saccharomyces cerevisiae) and in a nonrespiratory variant of S. cerevisiae grown under glucose limitation. 2. In baker's yeast phosphoenolpyruvate carboxykinase, hexosediphophatase and isocitrate lyase were completely repressed under anaerobic conditions. Their repression could be partially reversed by using intense aeration. 3. In the nonrespiratory variant these enzymes were absent independently of aeration. 4. Pyruvate carboxylase of baker's yeast showed maximal activity under anaerobic conditions. In the nonrespiratory variant pyruvate carboxylase had low activity under both anaerobic and aerobic conditions.     

Effects of Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by Metabolically Engineered Strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Enzymatic determination of biotin.

An enzymatic method for the quantitative determination of biotin has been developed. The method involves the enzymatic binding of biotin in situ to the pyruvate carboxylase apoprotein of biotin-deficient bakers' yeast and the subsequent estimation of the pyruvate carboxylase activity by a ¹⁴CO₂-fixation method. The method is specific for biotin. Several biotin analogs and precursors were tested, and only biocytin was found to interfere, Biotin amounts of less than 5 pg can be estimated.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.     

Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae

The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU·mg^-1 at this induced state. In glucoselimited, derepressed cells, 20 mU·mg^-1 were detected and under repressed conditions isocitrate lyase activity was not detected.The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6-bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%.     

Continuous cultivation of Saccharomyces cerevisiae at different biotin concentrations in nutrient media

An increase of biotin concentration in nutrient media increased the content of protein, phosphorus, total ribonucleic acids, activity of pyruvate carboxylase and isocitrate lyase in cells and decreased the content of trehalose, glycogen and respiratory quotient of yeast cells in the course of continuous cultivation of Saccharomyces cerevisiae.     

The control of the synthesis of isocitrate lyase in a thermophilic bacillus.

From a strain of Bacillus stearothermophilus, devoid of active pyruvate carboxylase, a mutant (NG-15) was selected that grew on acetate in the presence of glucose. This mutant differed from its parent organism in possessing high activities of isocitrate lyase when grown on all carbon sources tested except nutrient broth, in possessing unusually low activities of NADP+-dependent isocitrate dehydrogenase and in containing increased amounts of isocitrate. Revertants of mutant NG-15 which regained the ability to synthesize active pyruvate carboxylase also synthesized isocitrate lyase and isocitrate dehydrogenase to the same extent as the wild-type strain. These results suggest that the regulatory mechanism for the synthesis of isocitrate lyase in the thermophile may be different from that in mesophilic bacilli.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Physiological Role of Pyruvate Carboxylase in a Thermophilic Bacillus

A prototrophic, thermophilic bacillus is in a state of biotin insufficiency when grown in medium consisting of inorganic salts and a carbon source. The effect of this biotin deficiency on the growth rate is severe only if the functioning of pyruvate carboxylase is essential for the utilization of the particular growth substrate. A mutant, PC2, of the thermophile devoid of active pyruvate carboxylase has been isolated. The properties of this mutant confirm the anaplerotic role of this enzyme in the utilization for growth of compounds like glucose and lactate which are catabolized via pyruvate. This conclusion is supported by the finding that revertants isolated from strain PC2 have regained simultaneously the ability to synthesize active pyruvate carboxylase and the ability to utilize glucose or lactate for growth. The growth of mutant PC2 on acetate, unlike that of the parent wild type, is inhibited when glucose or lactate is added to the medium. Secondary mutants obtained from PC2, which are resistant to such inhibition, still carry the original pyruvate carboxylase lesion but are derepressed for isocitrate lyase. This suggests that the inhibition of the growth of mutant PC2 is due to a block in the functioning of the glyoxylate cycle, produced by the glucose or lactate supplement.     

Carboxylase Levels and Carbon Dioxide Fixation in Baker''s Yeast

Levels of pyruvate carboxylase (PC), phosphopyruvate carboxylase (PEPC), and malate dehydrogenase (decarboxylating) were compared in wild-type bakers' yeast (I), a cytoplasmic-respiratory mutant (II), a biotin-deficient wild-type yeast (III), and a biotin-deficient respiratory mutant (IV). PC activities were greatly reduced in III and IV, whereas PEPC was reduced in II and IV. Malate dehydrogenase (decarboxylating) could not be detected in any of the yeasts. With yeast I growing on glucose as the sole carbon source, PEPC decreased to negligible levels during the logarithmic phase of growth (glucose repression effect), whereas PC increased. Both enzymes reverted to their original levels during the stationary phase, when glucose in the medium was exhausted. In agreement with the leading role of PC for CO(2) assimilation, the rates of (14)CO(2) fixation in yeasts I and II were approximately equal and were much higher than that in yeast IV. With I and II, most of the (14)C was distributed similarly in oxalacetate derivatives; with yeast IV, most of (14)C appeared in a compound apparently unrelated to CO(2) fixation via C(4)-dicarboxylic acids.     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

Enzyme patterns during substrate shifts between phenol,acetate and glucose in continuous culture of Trichosporon cutaneum

Summary The soil yeast Trichosporon cutaneum was grown in continuous culture on phenol, acetate or glucose as sole carbon source. The activities of enzymes participating in the tricarboxylic acid cycle, glyoxylate cycle, 3-oxoadipate pathway, pentose phosphate pathway and glycolysis were determined in situ during shifts of carbon sources. Cells grown on phenol or glucose contained basal activity of the glyoxylate-cycle-specific isocitrate lyase. The derepression of the glyoxylate cycle enzymes was partly hindered in the presence of phenol but not in the presence of low levels of glucose. Phenol and glucose caused repression of isocitrate lyase. In the presence of either phenol or glucose, acetate accumulation in the medium increased. However, part of the supplied acetate was utilized simultaneously with phenol or glucose, the utilization rate of either carbon source being reduced in the presence of the other carbon source. Acetate caused repression but not inactivation of the phenol-degrading enzymes, phenol hydroxylase and catechol 1,2-dioxygenase. The simultaneous utilization of phenol and other carbon sources in continuous culture as well as the observed repression-derepression patterns of the involved enzymes reveal T. cutaneum to be an organism of interest for possible use in decontamination processes. Offprint requests to: H. Y. Neujahr Offprint requests to: H. Y. Neujahr     

Choline Dehydrogenase of Pseudomonas aeruginosa A-16

The promotive effect of biotin (200~500 µg/liter) on l-lysine formation was investigated in Brevibacterium lactofermentum. This effect was observed only when glucose or pyruvate was used as sole carbon source, and accompanied with the specific incorporation of ¹⁸CO₂ into γ-CH₂ group of l-lysine. Brev. lactofermentum AJ 3445 (AEC^r) could grow on pyruvate medium supplemented with biotin at more than 200 µg/liter, while the same growth was observed with the addition of TCA cycle members or glutamate to pyruvate medium.

Phosphoenolpyruvate (PEP) carboxylase deficient mutant derived from AJ 3445 could not grow on glucose as sole carbon source, but on glucose plus 200 µg/liter of biotin. AJ 3445 grown on lactate medium containing 500 µg/liter of biotin and KHCO₃ contained the biotin-dependent pyruvate carboxylase.

These data suggest that this promotive effect of excess biotin on l-lysine formation may be brought about through the activation of pyruvate carboxylase by biotin.     

Biotin uptake, utilization, and efflux in normal and biotin-deficient rat hepatocytes.

Biotin uptake, utilization, and efflux were studied in normal and biotin-deficient cultured rat hepatocytes. Biotin-deficient cells accumulate about 16-fold more biotin than do normal cells when incubated with a physiological concentration of biotin for 24 h. This difference is due to the greater amount of protein-bound biotin relative to free biotin in biotin-deficient hepatocytes, and is attributable to the presence of more apocarboxylases in deficient cells. The rate of biotin uptake and the rate of activation of the carboxylases, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase, are proportional to the concentration of exogenous biotin. Increases in carboxylase activities are proportional to the concentration of biotin only at exogenous biotin concentrations of less than 410 nM. Concentrations of 410 nM or more biotin increase carboxylase activities to normal or near normal. Biocytin inhibits biotin uptake at very high concentrations, whereas desthiobiotin and lipoic acid have no effect. Biocytin in the medium results in carboxylase activation either intracellularly or extracellularly by conversion to biotin by biotinidase. Investigation of the efflux of biotin from normal and biotin-deficient cells preincubated with the vitamin showed greater retention of biotin by biotin-deficient cells than by normal cells over 24 h. Retention of free biotin is similar in biotin-deficient and normal cells. The greater amount of biotin retained by biotin-deficient cells is accounted for by the greater amount of bound biotin in these cells. These results suggest that the free and bound biotin pools are independently regulated. The ready loss of free biotin from these cells has implications for the treatment of inherited, biotin-responsive carboxylase deficiencies.     

Exopolysaccharide Production and Peculiarities of C6-Metabolism in Acinetobacter sp. Grown on Carbohydrate Substrates

An Acinetobacter sp. strain grown on carbohydrate substrates (mono- and disaccharides, molasses, starch) was shown to synthesize exopolysaccharides (EPS). Glucose catabolism proved to proceed via the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. Pyruvate entered the tricarboxylic acid cycle due to pyruvate dehydrogenase activity. Pyruvate carboxylation by pyruvate carboxylase was the anaplerotic reaction providing for the synthesis of intermediates for the constructive metabolism of Acinetobacter sp. grown on C₆-substrates. The C₆-metabolism in Acinetobacter sp. was limited by coenzyme A. Irrespective of the carbohydrate growth substrate (glucose, ethanol), the activities of the key enzymes of both C₂- and C₆-metabolism was high, except for the isocitrate lyase activity in glucose-grown bacteria. Isocitrate lyase activity was induced by C₂-compounds (ethanol or acetate). After their addition to glucose-containing medium, both substrates were utilized simultaneously, and an increase was observed in the EPS synthesis, as well as in the EPS yield relative to biomass. The mechanisms responsible for enhancing the EPS synthesis in Acinetobacter sp. grown on a mixture of C₂- and C₆-substrates are discussed.     

Effect of growth condition on enzymes of the citric acid cycle and the glyoxylate cycle in the photosynthetic bacterium Rhodopseudomonas palustris

The enzymes of the citric acid and glyoxylate cycles as well as RuBP carboxylase were measured in cell-free extracts from Rhodopseudomonas palustris after growth under chemoheterotrophic, photoheterotrophic and photolithotrophic conditions. Although the citric acid cycle was found to be complete under all growth conditions, significant differences in certain enzyme activities occurred as a function of the different energy sources applied. The glyoxylate cycle also was complete under all growth conditions with highest isocitrate lyase activity seen after photoheterotrophic growth on acetate. Photo- and chemoheterotrophic growth on malate reduced the isocitrate lyase. The activity was not repressed further by photolithotrophic growth on thiosulfate. RuBP carboxylase activity, present under photolithotrophic conditions, was repressed by chemoheterotrophic growth but was not decreased by the presence of organic substrates during photoheterotrophic growth.

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Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype

We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.