OXIDATIVE METABOLISM AND THE GLYOXYLATE CYCLE IN PSEUDOMONAS INDIGOFERA.

McFadden, Bruce A. (Washington State University, Pullman, Wash.) and William V. Howes. Oxidative metabolism and the glyoxylate cycle in Pseudomonas indigofera. J. Bacteriol. 84:72-76. 1962.-Oxidative patterns of Pseudomonas indigofera have been investigated. Intact cells oxidize acetate, ethanol, fumarate, glyoxylate, alpha-ketoglutarate, malate, oxaloacetate, pyruvate, and succinate to greater than 35% of completion. Isocitrate is oxidized to 21% of completion. Citrate is not oxidized by whole cells but is oxidized by cell-free preparations, as are fumarate, isocitrate, malate, and succinate. These patterns are suggestive of the operation of the tricarboxylic acid cycle. Investigations of levels of isocitrate lyase and malate synthase as functions of growth substrate have been conducted. Assays for these enzymes in "soluble" preparations were performed under ostensibly optimal conditions for catalysis. Growth substrates used at 0.3% were: (i) ethanol, (ii) glucose, (iii) succinic acid, and (iv) yeast extract. Specific activities of isocitrate lyase were: for (i) 3.80, (ii) 0.61, (iii) 1.47, and (iv) 1.33; activities of malate synthase were: for (i) 0.18, (ii) 0.032, (iii) 0.021, and (iv) 0.029. Additionally, the isocitrate lyase level from butyrate-grown cells was similar to that for ethanol-grown cells; the specific activity of malate synthase was about 60% as high. Specific activities of these enzymes were reproducible when conditions of sonic disruption were standardized. Longer durations of disruption decreased both activities.     

Occurrence of Isocitrate Lyase in a Thermophilic Bacillus Species

A thermophilic, sporeforming bacterium has been isolated from soil on a medium containing acetate as a carbon source. This organism is similar to Bacillus stearothermophilus in most respects but differs in its inability to hydrolyze starch. Isocitrate lyase is present in cell-free extracts of organisms grown in a medium with acetate as a carbon source. The specific activity was 400 times lower in extracts of organisms utilizing glucose as a carbon source. With crude extracts, enzyme activity was strongly stimulated by Mg(++), but cysteine and ethylenediaminetetraacetate had little effect. It appeared to be more heat-stable than the pure isocitrate lyase from Pseudomonas indigofera.     

Inhibition of isocitrate lyase from Pseudomonas indigofera by itaconate

The effect of the inhibitor itaconate on the activity of purified isocitrate lyase from Pseudomonas indigofera was examined for the reaction in both directions. Itaconate was found to equilibrate very slowly with its enzyme-bound form, so that a rapid change in itaconate concentration produced a gradual change in reaction velocity which eventually reached a new steady state. Kinetic studies of this relaxation phenomenon indicated that itaconate inhibited by binding the enzyme only after prior binding of glyoxylate, thus mimicking the kinetic behavior of succinate. On the basis of these studies, the dissociation constants for itaconate and glyoxylate from their respective enzyme-bound forms were calculated. More than half of the isocitrate lyase was complexed by glyoxylate during cleavage of saturating isocitrate. The rate constant for release of itaconate from the enzyme was calculated to be about 0.2 min^?1. Direct binding of [¹⁴C]itaconate and [¹⁴C]succinate to isocitrate lyase at pH 6.8 was measured. Some binding of both ligands was found in the absence of glyoxylate, which was stimulated by the presence of 1 mm glyoxylate. These results suggest that there are up to three or more binding sites per active subunit, but that only one of these is catalytic.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

Inhibition of isocitrate lyase: the basis for inhibition of growth of two Arthrobacter species by pyruvate

Growth of Arthrobacter atrocyaneus and A. pyridinolis on certain growth substrates was found to be inhibited by pyruvate and compounds which can be converted to pyruvate. Growth of A. atrocyaneus on acetate, for example, was completely inhibited by 5 mm pyruvate; growth of this organism on glucose was less sensitive and growth on succinate was insensitive to inhibition by pyruvate. Growth of a third Arthrobacter species, A. crystallopoietes, on acetate and other substrates was not inhibited by pyruvate. The site of pyruvate inhibition was shown to be the isocitrate lyase reaction. Glyoxylate, which affords a bypass of this reaction, restored the ability of A. atrocyaneus to evolve (14)CO(2) from acetate in the presence of pyruvate. The isocitrate lyases from A. atrocyaneus and A. pyridinolis were competitively inhibited by concentrations of pyruvate as low as 1 mm, whereas the enzyme from A. crystallopoietes was unaffected by this concentration of pyruvate. Comparable levels of phosphoenolpyruvate did not inhibit the isocitrate lyases from any of the species. A mutant strain of A. atrocyaneus, PW11, which is deficient in isocitrate lyase activity, grew on glucose at a reduced rate that was comparable to the rate of growth of the wild-type strain on glucose plus lactate. Addition of lactate to PW11 did not further reduce its rate of growth on glucose. Thus, the glyoxylate pathway appears to be used as an anaplerotic pathway during growth of A. atrocyaneus on glucose. Two other considerations suggest that A. atrocyaneus and A. pyridinolis, but not A. crystallopoietes, may be deficient in the ability to convert pyruvate to 4-carbon acids. First, the former two species accumulate intracellular pyruvate from exogenous l-alanine to a much greater extent than does A. crystallopoietes. Moreover, A. atrocyaneus and A. pyridinolis are incapable of growth on lactate as sole source of carbon whereas A. crystallopoietes can grow on lactate.     

Inhibition by itaconate of growth of methylotrophic bacteria.

The effects of the isocitrate lyase-directed growth inhibitor itaconate on the growth of certain methylotrophic organisms was investigated. It was found that growth of those organisms possessing the Icl(+)-serine pathway of one-carbon metabolism was inhibited during growth on methylamine and on acetate, but not on glucose. Organisms possessing the Icl(-)-serine pathway pathway were unaffected. Organism PAR, an Icl(-)-serine pathway type, was not specifically inhibited during growth on acetate. This finding further substantiates previous reports of the lack of isocitrate lyase in this organism, indicating a totally new pathway for acetate assimilation.     

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.     

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.     

Enzyme Profiles in Seedling Development and the Effect of Itaconate, an Isocitrate Lyase-directed Reagent

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP⁺-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K_i:17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.     

Control of isocitrate lyase in Nocardia salmonicolor (NCIB9701).

Nocardia salmonicolor, grown on acetate, commercial D,L-lactate or hydrocarbon substrates, has high isocitrate lyase activities compared with those resulting from growth on other carbon sources. This presumably reflects the anaplerotic role of the glyoxylate cycle during growth on the former substrates. Amongst a variety of compounds tested, including glucose, pyruvate and tricarboxylic acid cycle intermediates, only succinate and fumarate prevented an increase in enzyme activity in the presence of acetate. When acetate (equimolar to the initial sugar concentration) was added to cultures growing on glucose, there followed de novo synthesis of isocitrated lyase and isocitrate dehydrogenase, with increases in growth rate and glucose utilization, and both acetate and glucose were metabolized simultaneously. A minute amount of acetate (40 muM) caused isocitrate lyase synthesis (a three-fold increase in activity within 3 min of addition) when added to glucose-limited continuous cultures, but even large amounts added to nitrogen-limited batch cultures were ineffective. Malonate, at a concentration that was not totally growth-inhibitory (1mM) prevented the inhibition of acetate-stimulated isocitrate lyase synthesis by succinate, but fumarate still inhibited in the presence of malonate. Phosphoenolpyruvate is a non-competitive inhibitor of the enzyme (apparent Ki 1-7 mM). The results are consistent with the induction of isocitrate or a closely related metabolite, and catabolite repression by a C-4 acid of the tricarboxylic acid cycle, possibly fumarate.     

Two-carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was grown on 10% glucose medium and subsequently transferred to fresh medium containing 2- and 3-carbon substrates. Under these conditions, the yeast rapidly acquired an oxidative capacity, as evidenced by oxygen uptake rates and 14CO2 evolution rates during respiration on ethanol or (14C)acetate. The assimilative capacity for 2-carbon substrates developed more slowly and followed the induction of isocitrate lyase. Washed yeast transferred to the basic medium containing no added carbon substrate possessed only low levels of isocitrate lyase after a 6-h adaptation. After 6 h, isocitrate lyase was present at high levels in cells transferred to a range of ethanol concentrations but was present in only low amounts in cells transferred to acetate. The role of ethanol as an inducer of isocitrate lyase is discussed.     

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.     

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.     

Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus.

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.     

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.     

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.     

Citrate Cycle and Related Metabolism of Listeria monocytogenes

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.     

Cell-free amino acid-incorporating system from Pseudomonas indigofera

Shiio, Tsuru (Washington State University, Pullman), and Bruce A. McFadden. Cell-free amino acid-incorporating system from Pseudomonas indigofera. J. Bacteriol. 90:978-983. 1965.-A cell-free preparation from Pseudomonas indigofera incorporated C(14)-phenylalanine and C(14)-leucine into a product which was insoluble in hot trichloroacetic acid. The phenylalanine incorporation process, which had a temperature optimum of 30 C and a pH optimum of 7.6, had many characteristics of protein synthesis. The process depended upon both "ribosomes" and supernatant fraction from centrifugation at 105,000 x g. Incorporation required adenosine triphosphate, apparently depended upon guanosine triphosphate, and was inhibited by chloramphenicol, puromycin, actinomycin, ribonuclease, and deoxyribonuclease. Leucine incorporation was also studied and had many similar characteristics. C(14)-phenylalanine uptake was stimulated by sRNA or polyuridylic acid, and together these substances had a synergistic effect upon stimulation. The incorporation of C(14)-phenylalanine into a product which was precipitated by antiserum to crystalline isocitrate lyase was also observed.