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.     

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.     

Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger

In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed.The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium.Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.     

Itaconate, an isocitrate lyase-directed inhibitor in Pseudomonas indigofera.

Enzymes catalyzing steps from ethanol to acetyl-coenzyme A, from malate to pyruvate, and from pyruvate to glucose 6-phosphate were identified in ethanol-grown Pseudomonas indigofera. Enzymes catalyzing the catabolism of glucose to pyruvate via the Entner-Doudoroff pathway were identified in glucose-grown cells. Phosphofructokinase could not be detected in Pseudomonas indigofera. Itaconate, a potent inhibitor of isocitrate lyase, abolished growth of P. indigofera on ethanol at concentrations that had little effect upon growth on glucose. The date obtained through enzyme analyses and studies of itaconate inhibition with both extracts and toluene-treated cells suggest that itaconate selectively inhibits and reduces the specific activity of isocitrate lyase.     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.     

Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two-fold in extracts of acetate grown cells.     

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.     

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.     

Mitochondrial enzymes in mango fruit during ripening

Mitochondria isolated from immature (developing), mature (unripe), and ripe mango pulp actively oxidized the intermediates of the Krebs cycle. The oxidation of citrate, oxoglutarate, succinate and malate by both unripe and ripe fruit mitochondria was several fold greater than that by mitochondria from immature fruit. The levels of malic dehydrogenase and succinic dehydrogenase increased with the onset of ripening, whereas the level of citrate synthase increased several fold on maturation but decreased six-fold on ripening. Isocitrate dehydrogenase and malic enzyme were very high in the immature fruit but after a sudden decrease in the matured fruit showed a considerable rise thereafter. The ratio of the activities of isocitrate lyase to isocitrate dehydrogenase is considerably higher in the immature fruit and greatest in the unripe (mature) fruit. This, together with a higher concentration of glyoxylate at these stages, indicate the operation of the glyoxylate bypass. Oxidized and reduced forms of pyridine nucleotides were estimated.     

Acetate metabolism in Rhodopseudomonas gelatinosa and several other Rhodospirillaceae

When Rhodopseudomonas gelatinosa was grown on acetate aerobically in the dark both enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, could be detected. However, under anaerobic conditions in the light only isocitrate lyase, but not malate synthase, could be found.The reactions, which bypass the malate synthase reaction are those catalyzed by alanine glyoxylate aminotransferase and the enzymes of the serine pathway.Other Rhodospirillaceae were tested for isocitrate lyase and malate synthase activity after growth with acetate; they could be divided into three groups: I. organisms possessing both enzymes; 2. organisms containing malate synthase only; 3. R. gelatinosa containing only isocitrate lyase when grown anaerobically in the light.     

The Glyoxylate Pathway in Acanthamoeba Sp.*

SYNOPSIS. Cell-free extracts of encysting Acanthamoeba were assayed for the key enzymes of the glyoxylate pathway, viz., isocitrate lyase and malate synthase. Both enzymes were present at the onset of encystment but their activities changed as cyst-wall formation proceeded to completion. Isocitrate lyase activity decreased during the first 4 hr of encystment to a minimum at 4 hr which was 70% of its initial activity. Activity then increased reaching a maximum at 9 hr which was 144% of its initial activity. After 9 hr a decrease in isocitrate lyase activity began which reached 70% of its initial activity at 35 hr. Malate synthase activity slowly decreased throughout encystment to 50% of its initial activity after 35 hr. From these data and others cited, it is concluded that this small soil amoeba has a functional glyoxylate pathway.     

Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.

Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (Ki = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 +/- 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer).     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

Effects of salts on aerobic metabolism of Debaryomyces hansenii

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.     

Regulation of Enzyme Synthesis of the Glyoxylate,the Citric Acid,and the Methylcitric Acid Cycles in Candida lipolytica

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.

In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-Iinked isocitrate dehydrogenase, isocitrate lyase, 2- methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-d_s-2-methylisocitric acid accumulation.     

Central metabolism in Acinetobacter sp. grown on ethanol

The ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The proportion between these enzymes was different in the exponential and the stationary growth phases. The addition of the C4-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of the microbial exopolysaccharide ethapolan on C2-substrates.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.