Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

A simple spectrophotometric assay for fumarate hydratase in crude tissue extracts

A procedure is described for assaying fumarate hydratase by coupling malate formed from fumarate to NADP⁺ reduction via NADP malic enzyme. The procedure is much more sensitive than existing assay methods and cireumvents problems particularly associated with the use of these methods for determining fumarate hydratase in crude tissue extracts.     

A spectrophotometric coupled enzyme assay to measure the activity of succinate dehydrogenase

Respiratory complex II (succinate:ubiquinone oxidoreductase) connects the tricarboxylic acid cycle to the electron transport chain in mitochondria and many prokaryotes. Complex II mutations have been linked to neurodegenerative diseases and metabolic defects in cancer. However, there is no convenient stoichiometric assay for the catalytic activity of complex II. Here, we present a simple, quantitative, real-time method to detect the production of fumarate from succinate by complex II that is easy to implement and applicable to the isolated enzyme, membrane preparations, and tissue homogenates. Our assay uses fumarate hydratase to convert fumarate to malate and uses oxaloacetate decarboxylating malic dehydrogenase to convert malate to pyruvate and to convert NADP⁺ to NADPH; the NADPH is detected spectrometrically. Simple protocols for the high-yield production of the two enzymes required are described; oxaloacetate decarboxylating malic dehydrogenase is also suitable for accurate determination of the activity of fumarate hydratase. Unlike existing spectrometric assay methods for complex II that rely on artificial electron acceptors (e.g., 2,6-dichlorophenolindophenol), our coupled assay is specific and stoichiometric (1:1 for succinate oxidation to NADPH formation), so it is suitable for comprehensive analyses of the catalysis and inhibition of succinate dehydrogenase activities in samples with both simple and complex compositions.     

PHOSPHOFRUCTOKINASE AND FUMARATE HYDRATASE IN DEVELOPING RAT BRAIN

The developmental patterns of phosphofructokinase, fumarate hydratase and lactate dehydrogenase were determined and compared using homogenates of rat brain. Phosphofructokinase activity, expressed in terms of tissue wet wt., was relatively constant from 5 days before birth to 8 days postnatal; a 110 per cent increase in activity occurred between 12 and 21 days of age, when adult levels were achieved. The degree of inhibition of phosphofructokinase by 1-0 mM-ATP changed little during development; inhibition by 2-5 mM-citrate was about 50 per cent in both newborn and adult brain. Phosphofructokinase development more closely resembled that of lactate dehydrogenase than that of fumarate hydratase.     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

Enzymatic synthesis of L-malic acid from fumaric acid using immobilized Escherichia coli cells

Optimal conditions were chosen for cultivation of Escherichia coli 85 cells with a rather high fumarate-hydratase activity on a cheap medium containing no edible raw material. An active biocatalyst for the synthesis of L-malic acid from fumaric acid was obtained based on E. coli 85 cells immobilized in carrageenan. The enzymatic synthesis of L-malic acid from potassium fumarate was kinetically studied and optimized. Some thermodynamic parameters of fumaric acid hydration into malic acid were determined. A technique for assaying the reaction mixture was developed that involved high performance liquid chromatography.     

NAD+-malic enzyme. Regulatory properties of the enzyme from Ascaris suum.

The regulatory properties of the NAD-dependent malic enzyme from the mitochondria of Ascaris suum have been studied. The malate saturation curve exhibits sigmoidicity and the degree of this sigmoidicity increases as the pH is increased. Fumarate was the only compound tested that stimulated the enzyme activity, whereas oxalacetate was the most powerful inhibitor. Activation by low levels of fumarate was found to be competitive with malate. It is proposed that this stimulation has physiological significance in controlling the dismutation reaction in the parasite. The branched-chain volatile fatty acid excretion products, tiglate, 2-methylbutanoate, and 2-methylpentanoate, inhibited the enzyme activity and this inhibition was competitive with malate. The Ki values for these compounds are in the physiological range of their concentrations; therefore, it is suggested that they may aid in controlling the malic enzyme activity in vivo. Oxalacetate inhibition of malic enzyme activity was competitive with malate, and the Ki values decreased with an increase in pH. Two alternatives are proposed which could account for the lack of oxalacetate decarboxylation by the ascarid malic enzyme.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Production of L-malate from fumarate by the yeast Dipodascus magnusii

An alternative microbiological method for the production of malate from fumarate is presented. The yeast Dipodascus magnusii was used for this bioconversion. The optimum cell growth temperature was 28°C and the working volume 120 ml. The highest level of fumarase activity during bioconversion was achieved at a pH of 7.5 and a temperature of 37°C. These conditions were determined as optimal. Using sodium fumarate (1M), the maximum specific productivity of malic acid obtained was 1.72 g/(g_DCW × h) for intact cells. In the case of ammonium fumarate, it was 2.25 g/(g_DCW × h).     

Biochemical Aspects of Fumaric Acid Accumulation by Rhizopus arrhizus

The accumulation and excretion of fumaric acid, and to a lesser extent malic and succinic acids, by Rhizopus arrhizus occurs under aerobic conditions in a high-glucose medium containing a limiting amount of nitrogen and a neutralizing agent (CaCO₃). An overall four-carbon dicarboxylic acid molar yield of up to 145% (moles of acid produced per mole of glucose utilized) is obtained after incubation for 4 to 5 days. Evidence is presented that fumarate is synthesized from pyruvate via a carboxylation reaction yielding oxaloacetate, which is then converted to malate and further on to fumarate via the reductive reactions of the tricarboxylic acid cycle. The possible formation of fumarate from the normal (oxidative) operation of the tricarboxylic acid cycle was not excluded by the data. Yield, ¹³C nuclear magnetic resonance, and enzymatic activity studies were carried out in a strain of R. arrhizus which produces high levels of fumarate from glucose and carbonate. The observed high fumarate molar yield (greater than 100%) can therefore be explained in terms of the carboxylation of pyruvate and the operation of the reductive reactions of the tricarboxylic acid cycle under aerobic conditions.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

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.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate.

Aspartate availability was increased in Corynebacterium glutamicum strains to assess its influence on lysine production. Upon addition of fumarate to a strain with a feedback-resistant aspartate kinase, the lysine yield increased from 20 to 30 mM. This increase was accompanied by the excretion of malate and succinate. In this strain, fumaric acid was converted to aspartate by fumarate hydratase, malate dehydrogenase, and aspartate amino transferase activity. To achieve the direct conversion of fumarate to aspartate, shuttle vectors containing the aspA+ (aspartase) gene of Escherichia coli were constructed. These constructions were introduced into C. glutamicum, which was originally devoid of the enzyme aspartase. This resulted in an aspartase activity of 0.3 U/mg (70% of the aspartase activity in E. coli) with plasmid pZ1-9 and an activity of up to 1.05 U/mg with plasmid pCE1 delta. In aspA+-expressing strains, lysine excretion was further increased by 20%. Additionally, in strains harboring pCE1 delta, up to 27 mM aspartate was excreted. This indicates that undetermined limitations in the sequence of reactions from aspartate to lysine exist in C. glutamicum.     

The tricarboxylic and acid pathway in Desulfovibrio.

Strains of two species of Desulfovibrio were examined for enzymes of the tricarboxylic acid cycle and related pathways. Pyruvate carboxylase (EC6.4.1.1) is present, and alpha-ketoglutarate is formed via the tricarboxylic acids. Glutamate, but not succinyl-CoA, arises from alpha-ketoglutarate. A pathway exists from pyruvate by malic enzyme (EC 1.1.1.39) activity to malate, then fumarate and succinate, again with no evidence of succinyl-CoA formation. The enzymes concerned with metabolism of these dicarboxylic acids show greater activity in the strains that can grow by fumarate dismutation. Glutamate (or glutamine), alpha-ketoglutarate, and yeast extract repress the enzymes that metabolize the tricarboxylic acids. There appears to be no glyoxylate cycle in Desulfovibrio vulgaris or D. desulfuricans.     

Organic acid metabolism in Sclerotinia sclerotiorum

Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.     

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.     

Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli.

The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells.     

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.