Metabolic Role, Regulation of Synthesis, Cellular Localization, and Genetic Control of the Glyoxylate Cycle Enzymes in Neurospora crassa

The glyoxylate shunt enzymes, isocitrate lyase and malate synthase, were present at high levels in mycelium grown on acetate as sole source of carbon, compared with mycelium grown on sucrose medium. The glyoxylate shunt activities were also elevated in mycelium grown on glutamate or Casamino Acids as sole source of carbon, and in amino acid-requiring auxotrophic mutants grown in sucrose medium containing limiting amounts of their required amino acid. Under conditions of enhanced catabolite repression in mutants grown in sucrose medium but starved of Krebs cycle intermediates, isocitrate lyase and malate synthase levels were derepressed compared with the levels in wild type grown on sucrose medium. This derepression did not occur in related mutants in which Krebs cycle intermediates were limiting growth but catabolite repression was not enhanced. No Krebs cycle intermediate tested produced an efficient repression of isocitrate lyase activity in acetate medium. Of the two forms of isocitrate lyase in Neurospora, isocitrate lyase-1 constituted over 80% of the isocitrate lyase activity in acetate-grown wild type and also in each of the cases already outlined in which the glyoxylate shunt activities were elevated on sucrose medium. On the basis of these results, it is concluded that the synthesis of isocitrate lyase-1 and malate synthase in Neurospora is regulated by a glycolytic intermediate or derivative. Our data suggest that isocitrate lyase-1 and isocitrate lyase-2 are the products of different structural genes. The metabolic roles of the two forms of isocitrate lyase and of the glyoxylate cycle are discussed on the basis of their metabolic control and intracellular localization.     

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus,an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min^-1·mg^-1 protein in thiosulphate-grown cells to 58 nmol·min^-1·mg^-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.Abbreviations TCA tricarboxylic acid - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - APAD acetylpyridine adenine dinucleotide - PMS phenazine methosulphate - DCPIP 2,6-dichlorophenol-indophenol - DOC dissolved organic carbon     

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.     

Isolation and biochemical properties of two types of microbody from Neurospora crassa cells.

Cells of the Neurospora crassa slime mutant grown in sucrose medium exhibited low activities of glyoxysomal marker enzymes isocitrate lyase (ICL), malate synthetase (MS), and malate dehydrogenase. Transfer of the cells to a medium containing acetate as sole carbon source ("acetate medium") induced a strong increase in the activities of these enzymes in both the soluble and the crude particulate cell fraction. Soluble isocitrate lyase activity increased rapidly after a lag phase of about 45 minutes. Addition of 0.1 mM cycloheximide to the acetate medium 3 hours after transfer of the cells halted the rise of isocitrate lyase activity in either cell fraction, but the inhibition of the incorporation of ICL activity into the particulate cell fraction was delayed by 1 hour. Addition of 20 g/l glucose resulted in the immediate decrease of both soluble and particulate ICL activities. Transfer to acetate medium induced no change in the activities of other microbody marker enzymes such as catalase, uricase or D-amino acid oxidase. Resolution of crude homogenates of "slime" cells by sucrose density gradient centrifugation yielded two major protein bands: A mitochondrial band at a density of 1.180 kg/l showing maximum activites of fumarase, isocitrate dehydrogenase and cytochrome c oxidase, and a microbody-rich band which obviously consisted of two types of organelles with different biochemical properties. Maximum activities of ICL and MS sedimented at a density of 1.21 kg/l while the peaks of particulate uricase and catalase activities were recovered at 1.24 kg/l.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Regulation of glyoxylate cycle enzymes in Saccharomycopsis lipolytica. I. Effect of the carbon source on isocitrate lyase and malate synthase activity

Comparative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximide and actinomycin D. The results were discussed comparing them with data obtained from other organisms.     

Glyoxylate metabolism in growth and sporulation of Bacillus cereus

Megraw, Robert E. (Iowa State University, Ames), and Russell J. Beers. Glyoxylate metabolism in growth and sporulation of Bacillus cereus. J. Bacteriol. 87:1087-1093. 1964.-Isocitrate lyase and malate synthetase were found in cell-free extracts of Bacillus cereus T. The patterns of synthesis of enzymes of the glyoxylic acid cycle were dependent upon the medium in which the organism was grown. Cells grown in acetate or in an acetate precursor, such as glucose, produced enzymes of the glyoxylic acid cycle in greatly diminished quantities, as compared with cells grown in media containing glutamate or yeast extract as principal carbon sources. Glutamate-grown cells had high isocitrate lyase activity but very low malate synthetase activity. Glyoxylate produced in this situation is metabolized by alternate pathways: conversion to tartronic semialdehyde and the latter to glyceric acid, thus providing evidence for a glycerate pathway; and reduction to glycolate (the reverse of this reaction was present at a low rate). Enzymatic activity of the glyoxylic acid cycle declines at the point where sporogenesis begins, indicating a metabolic shift for the synthesis of spore material.     

Taurine catabolism: III. Evidence for the participation of the glyoxylate cycle

The metabolic fate of acetate, produced during taurine catabolism in Pseudomonas aeruginosa TAU-5, appear to involve the glyoxylate cycle. Organisms grown on taurine have significantly higher levels of malate synthetase and isocitrate lyase than cells grown on nutrient broth, but were comparable to the levels found in acetate-grown organisms. Itaconate, an isocitrate lyase inhibitor, produced a prolonged lag phase and reduced the growth rate of organisms when it was present in the taurine or acetate growth medium. Ethylmethanesulfonate treatment of TAU-5 yielded mutant strains unable to grow on taurine or acetate as sole carbon sources, due to a lack of either malate synthetase or isocitrate lyase. Spontaneous revertants derived from these mutant strains regained the missing enzyme activity and the ability to grow on taurine or acetate.     

Taurine catabolism. III. Evidence for the participation of the glyoxylate cycle

The metabolic fate of acetate, produced during taurine catabolism in Pseudomonas aeruginosa TAU-5, appears to involve the glyoxylate cycle. Organisms grown on taurine have significantly higher levels of malate synthetase and isocritrate lyase than cells grown on nutrient broth, but were comparable to the levels found in acetate-grown organisms. Itaconate, an isocitrate lyase inhibitor, produced a prolonged lag phase and reduced the growth rate of organisms when it was present in the taurine or acetate growth medium. Ethylmethanesulfonate treatment of TAU-5 yielded mutant strains unable to grow on taurine or acetate as sole carbon sources, due to a lack of either malate synthetase or isocitrate lyase. Spontaneous revertants derived from these mutant strains regained the missing enzyme activity and the ability to grow on taurine or acetate.     

Intracellular Localization of Several Enzymes in Candida tropicalis Grown on Different Carbon Sources

Intracellular localization of several enzymes related to tricarboxylic acid cycle was investigated during the aerobic growth of Candida tropicalis on acetate, n-alkane and glucose. NADP-linked isocitrate dehydrogenase in acetate-grown cells was mostly found in S₂ fraction (20,000 × g supernatant fraction of protoplast lysate), whereas more than half of this activity in n-alkane-grown cells was recovered in P₂ fraction (20,000 × g pellet fraction). Large parts of NAD-linked isocitrate dehydrogenase and malate dehydrogenase were present in P₂ fraction, while NADP- and NAD-linked glutamate dehydrogenases were found preferentially in S₂ fraction, irrespective of the growth substrates used. Isocitrate lyase was detected in both fractions. Citrate synthase and aconitase in acetate-grown cells were almost particulate. Catalase activity recovered in P₂ fraction was far higher in alkane-grown cells than in acetate- or glucose-grown cells.     

Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.     

Evidence for the presence of the glyoxylate cycle in Chloroflexus

The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were present in cell-free extracts of the phototrophic, green, thermophilic bacterium Chloroflexus aurantiacus grown with acetate as the sole organic carbon source.The optimum temperature of these enzymes was 40° C, and their specific activities were high enough to account for the observed growth rate. Lower levels of the enzymes were found in extracts from cells grown on a complete medium.Itaconate was shown to inhibit isocitrate lyase from C. aurantiacus 96% at a concentration of 0.25 mM and also had a profound effect on the growth of the organism on acetate, 0.25 mM inhibiting completely. Itaconate also inhibited the growth when added to the complex medium, but in this case much higher concentrations were required.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

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.     

Metabolism of C2 compounds inAcetobacter aceti

The presence of isocitrate lyase and malate synthase was detected in cell-free extracts ofAcetobacter aceti, grown in a mineral medium with acetate as sole carbon source. The presence of these enzymes explains the ability of this strain to grow with ethanol or acetate as sole carbon source, which is an important characteristic in Frateur's classification system forAcetobacter. In addition to isocitrate lyase and malate synthase, these cell-free extracts were found to contain glyoxylate carboligase, tartronicsemialdehyde reductase and glycerate kinase. The induction of these enzymes during growth on acetate is thought to be caused by the very high activity of isocitrate lyase, which may lead to an accumulation of glyoxylate. The importance of this pathway in cells growing with acetate as sole carbon source for the synthesis of their carbohydrate components is discussed. The presence of the enzymes from the pathway from glyoxylate to 3-phosphoglycerate explains the ability of this strain to grow with ethyleneglycol and glycollate as sole carbon source.     

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.     

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     

Microbody-marker Enzymes during Transition from Phototrophic to Organotrophic Growth in Euglena

Transfer of Euglena gracilis Klebs Z cells from phototrophic to organotrophic growth on acetate results in derepression of the key enzymes of the glyoxylate cycle, malate synthase and isocitrate lyase, which appear coordinately regulated. The derepression of malate synthase and isocitrate lyase was accompanied by increased specific activities of succinate dehydrogenase, fumarase, and malate dehydrogenase, but hydroxypyruvate reductase activity was unaltered.