Partial Purification of Isocitrate Lyase from Candida tropicalis and Some Kinetic Properties of the Enzyme

Isocitrate lyase was purified partially from n-alkane-grown cells and glucose-grown cells of Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. The preparation from alkane-grown cells showed one peak of the enzyme activity, while that from glucose-grown cells showed two distinct peaks of the activity, on DEAE-cellulose column chromatography. These enzymes, having the similar pH optima (around 7.0) and Km values with dl-isocitrate (1.2 ~ 1.7 mm), were inhibited by various metabolic intermediates, such as 6-phosphogluconate and phosphoenolpyruvate.

Time-course changes in the activities of isocitrate lyase and isocitrate dehydrogenases of C. tropicalis during the growth indicated that the lyase would participate preferentially in alkane assimilation and NAD-linked isocitrate dehydrogenase in glucose utilization of the yeast.

Regulation of isocitrate metabolism in C. tropicalis through glyoxylate cycle and tricarboxylic acid cycle is discussed based on the kinetic properties, cellular localization and time- course changes in the levels of isocitrate lyase and NAD-linked and NADP-linked isocitrate dehydrogenases.     

Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast,Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Peroxisomal NADP-linked isocitrate dehydrogenase (Ps-NADP-IDH) was purified for the first time from Candida tropicalis cells grown on n-alkane as a carbon source, which was effective in proliferation of peroxisomes. The properties of Ps-NADP-IDH were compared with those of mitochondrial NAD-linked isocitrate dehydrogenase (Mt-NAD-IDH) purified from the cells grown on acetate, in which peroxisomes did not proliferate. Ps-NADP-IDH was a homodimer of identical subunits (45 kDa), while Mt-NAD-IDH was suggested to be a heterooctamer composed of two types of subunits with different molecular masses (41 and 38 kDa). Kinetic studies revealed that Ps-NADP-IDH gave Michaelis-Menten saturation curves against isocitrate and NADP concentrations, whereas Mt-NAD-IDH was an allosteric enzyme regulated by ATP, AMP, and citrate. Inhibition by 2-oxoglutarate, a precursor of glutamate, was observed only for Ps-NADP-IDH. Both enzymes were inhibited by concomitant addition of oxalacetate and glyoxylate. The function of Ps-NADP-IDH seems to be completely discriminated from that of Mt-NAD-IDH as reflected by their distinct subcellular localizations. Furthermore, the properties of Ps-NADP-IDH were also compared with those of other mitochondrial and cytosolic IDHs from sources reported previously.     

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.     

Some Characteristics of Central Metabolism in Acinetobacter sp. Grown on Ethanol

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 ratio of 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 microbial exopolysaccharide ethapolan on C₂-substrates.     

Intensification of surfactant synthesis in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP⁺-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0^?5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.     

Studies on Plant Growth-regulating Activities of Anisomycin and Toyocamycin

A new enzyme, 2-methylisocitrate dehydratase, isolated from Yallowia lipolytica, functioning in the methylcitric acid cycle for propionate metabolism, had a pI of 4.4 and a M_r of 69,500. The enzyme was composed of 624 residues of amino acids per molecule. No cofactor was required for full enzyme activity. The enzyme was competitively inhibited by threo-D_s-isocitrate (K_i =68mM), but not by any other tested metabolites. The enzyme was weakly inhibited by some thiol reagents, but not by any metal-chelating reagents, differing from aconitase, which dehydrates 2-methylisocitrate. This difference between the enzymes made it possible to estimate the activity of the new enzyme even in crude cell-free extracts. The enzyme was constitutively synthesized, but had no regulatory function in the methylcitric acid cycle. The enzyme was supposed to have evolutionarily developed from a hypothetical and prototypical isocitrate dehydratase.     

Effects of Chain-length of Alkane Substrate on Fatty Acid Composition and Biosynthetic Pathway in Some Candida Yeasts

Cellular fatty acid compositions of Candida tropicalis pK 233 and Candida lipolytica NRRL Y -6795 and the time-course changes during yeast growth were studied using individual n-alkanes of various chain lengths (from C₁₁ to C₁₈) and a mixture of n-alkanes (C₁₁ to C₁₈) as a sole carbon source. Observed relationships of the chain-length of n-alkane substrate to time-course changes and final patterns of the fatty acid compositions of these yeasts, especially those of the cells grown on odd-carbon alkanes, indicated that “intact incorporation mechanism,” that is, accumulation of the fatty acid having the same chain-length as that of the alkane substrate used was predominant in the yeasts cultivated on a longer alkane such as n-heptadecane and n-octadecane. On the other hand, “chain elongation pathway” and “de novo synthesis pathway” following β-oxidation of substrate were simultaneously operative in the cells growing on a relatively shorter alkane such as undecane and dodecane.     

Control of the citric acid cycle by glyoxylate: Mechanism of the inhibition by oxalomalate and γ-hydroxy-α-oxoglutarate

1. Hydroxyoxoglutarate was obtained by three methods: decarboxylation of oxalomalic acid, and synthesis from glyoxylate and pyruvate by using either Mg²⁺ or an enzyme from rat liver as catalysts. 2. The inhibitory effects of oxalomalate and hydroxyoxoglutarate upon aconitate hydratase, isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase were investigated. 3. Oxalomalate at low concentrations (1mm) inhibited almost completely both aconitate hydratase and isocitrate dehydrogenase. Hydroxyoxoglutarate also inhibited these enzymes, but at concentrations approximately tenfold that of oxalomalate. 4. Oxalomalate and hydroxyoxoglutarate, at the higher concentrations, inhibited oxoglutarate dehydrogenase to approximately the same extent. 5. It is suggested that the ability of glyoxylate to control reaction rates in the tricarboxylic acid cycle must in some degree be due to its condensation with oxaloacetate and pyruvate to form enzyme inhibitors.     

Microbody of n-alkane-grown yeast

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and d-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, malate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 nor NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.     

Effects of Carbon and Nitrogen Sources on the Levels of Several NADP- and NAD-Linked Dehydrogenase Activities of Hydrocarbon-utilizable Candida Yeasts

The variation of activities of several NADP-linked and NAD-linked dehydrogenases were studied during the aerobic growth of two species of hydrocarbon-utilizable Candida yeasts on different carbon and nitrogen sources. The level of NADP-linked isocitrate dehydrogenase in C. tropicalis and C. lipolytica growing on acetate was significantly higher than that in the yeasts growing on glucose. The glucose-grown cells of C. tropicalis showed a high activity of glucose-6-phosphate dehydrogenase as compared with the acetate-grown cells, while the enzyme level in C. lipolytica was low regardless of carbon sources used. The cells of both yeasts growing on n-alkane and oleic acid contained relatively low activity of NADP-linked isocitrate dehydrogenase. Presence of ion in the acetate medium increased the level of NADP-linked isocitrate dehydrogenase activity. These results suggest that different types of NADPH-generating systems operate alternatively in these yeasts depending upon carbon and nitrogen sources.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C₁₀H₂₂) to tetracontane (C₄₀H₈₂) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.     

Thermostability of enzymes of the tricarboxylic acid cycle ofBacillus coagulans

The thermostability of four enzymes of the tricarboxylic acid cycle has been studied in the facultative thermophile,Bacillus coagulans. Although isocitrate dehydrogenase appeared to be more temperature-sensitive in whole-cell extracts of cultures grown at 30°C compared with that in cultures grown at 55°C, this difference could be largely eliminated by the removal of cell-wall material. The specific activity of each of the enzymes examined was approximately threefold higher in cultures grown at 55°C than in those grown at 30°C. The maximum temperature, Arrhenius plot and effect of stabilizing agents for each enzyme were examined and found to be independent of growth temperature. Sodium chloride (10% w/v) was an effective protective agent for fumarase, aconitase and malate dehydrogenase. Protection from thermal denaturation of isocitrate dehydrogenase, aconitase and fumarase but not malate dehydrogenase was also given when the enzymes were heated in the presence of their substrates. These results are discussed in light of the generalized theories of facultative thermophily which have been proposed.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

2-Methylcitrate Dehydratase,a New Enzyme Functioning at the Methylcitric Acid Cycle of Propionate Metabolism

2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase), a new enzyme functioning at the methylcitric acid cycle of propionyl-CoA oxidation, was present in the cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. The enzyme was separated from the usual aconitate hydratase (EC 4.2.1.3) of the yeast with DEAE-Sephadex A-50 column chromatography. The enzyme was able to catalyze a reversible reaction between 2-methylcitrate and 2-methyl-cis-aconitate, but showed no activity on threo-d_s-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-d_s-, threo-DL- or erythro-l_s-isocitrate, DL-homocitrate or other hydroxy-acids tested.

In contrast, the other enzyme fraction separated as aconitate hydratase by chromatography showed no activity on synthetic 2-methylcitrate, but was able to catalyze strongly a reversible reaction between 2-methyl-cis-aconitate and threo-d_s-2-methylisocitrate.

From these findings, the previously proposed cycle sequence was revised at the following broken arrows: propionyl-CoA+oxaloacetate → (CoASH+) 2-methylcitrate ? 2-methyl-cis-aconitate ? threo-d_s-2-methylisocitrate → pyruvate+succinate (→→oxaloacetate).

2-Methylcitrate dehydratase showed maximum activity at pH 6.5 to 7.0 and at 25 to 40°C. The enzyme was stable at temperatures up to 40°C and at pH 6.5 to 7.5, but labile in Tris-HCl buffer. The synthesis of this enzyme was constitutive in this yeast, although it was slightly repressed by glucose.     

Effect of Metabolites of γ-Aminobutyric Shunt on Activities of NAD- and NADP-Isocitrate Dehydrogenases and Aconitate Hydratase from Higher Plants

We studied the influence of metabolites of -aminobutyric shunt of the tricarboxylic acid cycle on the activities of aconitate hydratase (EC.4.2.1.3) as well as NAD- and NADP-specific isocitrate dehydrogenases (EC.1.1.1.41 and EC.1.1.1.42, respectively) using purified enzyme preparations from pea leaves (Pisum sativum L.) and maize scutellum (Zea mays L.). -Aminobutyric acid and succinate proved to have no significant effect on these enzymes, while 0.1–0.2 mM glutamate markedly activated NADP isocitrate dehydrogenase from both sources, especially at unsaturating concentrations of the substrate. Succinic semialdehyde stimulated the activities of aconitate hydratase and NADP isocitrate dehydrogenase. The obtained data point to a similar pattern of the effect of intermediates of -aminobutyric shunt on the studied enzymatic activities for both photosynthetic tissues (pea leaves) and those with acidifying, transport, and digestive functions (maize scutellum). However, the absence of pronounced control effects of most metabolites on the studied enzymes allows us to assign them to a relatively inert pool of metabolites.     

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.     

NADPH/NADP+ ratio: regulatory implications in yeast glyoxylic acid cycle

Summary The utilization by yeast of two carbon sources is carried out through the operation of the glyoxylic acid cycle. Kinetic data from the isocitrate transforming enzymes suggest that the flow of isocitrate through the glyoxylic acid cycle depends upon the inhibition of the isocitrate decarboxylating enzymes. Both isocitrate dehydrogenases are inhibited by a mixture of glyoxylate + oxaloacetate, but for the reasons described in the text we consider that this inhibition is of no physiological significance. On the other hand, we have found that NADPH is a competitive inhibitor of NADP-isocitrate dehydrogenase with respect to NADP⁺, with a K_I similar to its K_M. It also produces an additive effect on the NADH-produced inhibition of NAD-isocitrate dehydrogenase. We propose NADPH as the compound that channels the utilization of isocitrate into the glyoxylic acid cycle. This is supported by the finding of an increased NADPH/NADP⁺ ratio in acetate grown yeast with respect to glucose grown cells.