Oxalate accumulation from citrate by Aspergillus niger. I. Biosynthesis of oxalate from its ultimate precursor.

Carbon-14 was incorporated from citrate-1,5-14C, glyoxylate-14C(U), or glyoxylate-1-14C into oxalate by cultures of Aspergillus niger pregrown on a medium with glucose as the sole source of carbon. Glyoxylate-14C(U) was superior to glyoxylate-1-14C and citrate-1,5-14C as a source of incorporation. By addition of a great amount of citrate the accumulation of oxalate was accelerated and its maximum yield increased. In a cell-free extract from mycelium forming oxalate from citrate the enzyme oxaloacetate hydrolase (EC3.7.1.1) was identified. Its in vitro activity per flask exceeded the rate of in vivo accumulation of oxalate. Glyoxylate oxidizing enzymes (glycolate oxidase, EC1.1.3.1; glyoxylate oxidase, EC1.2.3.5;NAD(P)-dependent glyoxylate dehydrogenase; glyoxylate dehydrogenase, CoA-oxalylating, EC1.2.1.7) could not be detected in cell-free extracts. It is concluded that in cultures accumulating oxalate from citrate after pregrowth on glucose, oxalate arises by hydrolytic cleavage of oxaloacetate but not by oxidation of glyoxylate.     

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.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Oxalate- and Glyoxylate-Dependent Growth and Acetogenesis by Clostridium thermoaceticum

The acetogenic bacterium Clostridium thermoaceticum ATCC 39073 grew at the expense of the two-carbon substrates oxalate and glyoxylate. Other two-carbon substrates (acetaldehyde, acetate, ethanol, ethylene glycol, glycolaldehyde, glycolate, and glyoxal) were not growth supportive. Growth increased linearly with increasing substrate concentrations up to 45 mM oxalate and glyoxylate, and supplemental CO₂ was not required for growth. Oxalate and glyoxylate yielded 4.9 and 9.4 g, respectively, of cell biomass (dry weight) per mol of substrate utilized. Acetate was the major reduced end product recovered from oxalate and glyoxylate cultures. ¹⁴C labeling studies showed that oxalate was subject to decarboxylation, and product analysis indicated that oxalate was utilized by the following reaction: 4^-OOC-COO^- + 5H₂O → CH₃COO^- + 6HCO₃^- + OH^-. Oxalate- and glyoxylate-dependent growth produced lower acetate concentrations per unit of cell biomass synthesized than did H₂-, CO-, methanol-, formate-, O-methyl-, or glucose-dependent growth. Protein profiles of oxalate-grown cells were dissimilar from protein profiles of glyoxylate-, CO-, or formate-grown cells, suggesting induction of new proteins for the utilization of oxalate. C. thermoaceticum DSM 2955 and Clostridium thermoautotrophicum JW 701/3 also grew at the expense of oxalate and glyoxylate. However, oxalate and glyoxylate did not support the growth of C. thermoaceticum OMD (a nonautotrophic strain) or six other species of acetogenic bacteria tested.     

Oxalate synthesis from [14C1]glycollate and [14C1]glycoxylate in the hepatectomized rat

Hepatectomy significantly altered the metabolism of [1-¹⁴C]glyoxylate and [1-¹⁴C]glycollate in the rat. The production of ¹⁴CO₂ was reduced by 47% and 77%–86%, respectively, indicating the involvement of the liver in the oxidation of both substrates. Unidentified intermediates, assumed to be primary glycine, serine and ethanolamine, were also reduced by over 50%, was would be expected from the removal of the aminotransferase enzymes through the hepatectomy. The biosynthesis of [¹⁴C]oxalate from [1-¹⁴C]glycollate was reduced by more than 80% in the hepatectomized rat. This suggests that this oxidation is primarily catalyzed by the liver enzymes, glycolic acid oxidase and glycolic acid dehydrogenase, in the intact rat. The limited formation of [¹⁴C]oxalate from [¹⁴₁]glycollate observed in the hepatectomized rat is probably catalyzed by lactate dehydrogenase or extrahepatic glycolic acid oxidase. Hepatectomy did not significantly alter the rate of formation of [¹⁴C]oxalate from [¹⁴₁]glyoxylate. However, since saturating concentrations of glyoxylate could not be used because of the toxicity of this substrate, the involvement of glycollic acid oxidase in this oxidation reaction in the intact rat can not be ruled out. In the hepatectomized rat, lactate dehydrogenase appears to be the enzyme making the major contribution, although other as yet not identified enzymes may be contributing. The increased deposition of oxalate in the tissues, oxalosis, may result from the shift in oxalate synthesis from the liver to the extrahepatic tissues.     

The metabolism of glyoxylate by cell-free extracts of Pseudomonas sp

1. Extracts of Pseudomonas sp. grown on butane-2,3-diol oxidized glyoxylate to carbon dioxide, some of the glyoxylate being reduced to glycollate in the process. The oxidation of malate and isocitrate, but not the oxidation of pyruvate, can be coupled to the reduction of glyoxylate to glycollate by the extracts. 2. Extracts of cells grown on butane-2,3-diol decarboxylated oxaloacetate to pyruvate, which was then converted aerobically or anaerobically into lactate, acetyl-coenzyme A and carbon dioxide. The extracts could also convert pyruvate into alanine. However, pyruvate is not an intermediate in the metabolism of glyoxylate since no lactate or alanine could be detected in the reaction products and no labelled pyruvate could be obtained when extracts were incubated with [1-¹⁴C]glyoxylate. 3. The ¹⁴C was incorporated from [1-¹⁴C]glyoxylate by cell-free extracts into carbon dioxide, glycollate, glycine, glutamate and, in trace amounts, into malate, isocitrate and α-oxoglutarate. The ¹⁴C was initially incorporated into isocitrate at the same rate as into glycine. 4. The rate of glyoxylate utilization was increased by the addition of succinate, α-oxoglutarate or citrate, and in each case α-oxoglutarate became labelled. 5. The results are consistent with the suggestion that the carbon dioxide arises by the oxidation of glyoxylate via reactions catalysed respectively by isocitratase, isocitrate dehydrogenase and α-oxoglutarate dehydrogenase.     

The role of citrate in lipid synthesis in developing soybean cotyledons

The role of citrate and the citrate cleavage enzyme in lipidsynthesis in developing soybean cotyledons (Glycine max L. Merr.var. Harosoy 63) was investigated. The activity of the enzymewas inhibited by (—) hydroxycitrate, which is a specificinhibitor of citrate cleavage by this enzyme. Incorporationof label from citrate-1-¹⁴C and -5-¹⁴C indicated that the citratemolecule is cleaved between carbons 3 and 4. Acetyl CoA-¹⁴Cand oxaloacetate-¹⁴C phenylhydrazone were isolated as productsof the citrate cleavage reaction. The production of oxaloacetate-¹⁴C-phenylhdrazonefrom citrate-6-¹⁴C was carried out using a nucleotide free enzymepreparation and did not require the addition of ATP or CoA.Therefore it would appear that the citrate cleavage reactionis not CoA dependent in developing soybean seeds. Incorporationof pyruvate-2-¹⁴C into the crude lipid fraction was shown torequire both the particulate and soluble fractions. Apparentlyin soybeans, as in animal systems, pyruvate is oxidized by thepyruvate dehydrogenase complex and the acetyl CoA formed condenseswith oxaloacetate to produce citrate in the mitochondria. Citrateis then transported out of the mitochondria to the cytosol whereit is cleaved to form acetyl CoA for lipid synthesis. ¹ Cooperative investigations of the Agricultural Research Service,U.S. Department of Agriculture, and Illinois Agricultural ExperimentStation. ² This research represents partial fulfillment of the Ph. D.requirements of Daniel R. Nelson. Presently at Monsanto AgriculturalProducts Co., St. Louis, MO 63141, U.S.A. (Received January 12, 1977; )     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

The biosynthesis of trichothecin from acetate- [1,2- 13C2]

The coupling pattern of trichothecin biosynthesized from acetate-[1,2-¹³C₂] is in accord with previous enrichment studies. Multiple labelling was observed. Exogenous acetate has been shown to inhibit the utilization of glucose and the incorporation of radioactivity from pyruvate-[2-¹⁴C] and citrate-[1,5-¹⁴C] into the metabolites. Two pairs of ¹³C NMR assignments are interchanged.     

Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays

CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-¹⁴C]citrate formed in the presence of [4-¹⁴C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-¹⁴C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE dithioerythritol - NEM N-ethylmaleimide - NES N-ethylsuccinimide Enzymes (EC 2.7.2.1) Acetate kinase, ATP: acetate phosphotransferase - (EC 3.1.3.1) Alkaline phosphatase, orthophosphoric monoester phosphohydrolase - (GOT) Aspartate aminotransferase - (EC 2.6.1.1) L-aspartate:2-oxoglutarate aminotransferase - (CS) Citrate synthase - (EC 4.1.3.7) citrate oxaloacetate-lyase (pro 3S-CH₂COOacetyl-CoA) - (EC 2.8.3.8) CoA-transferase, acyl-CoA:acetate CoA-transferase - (EC 1.1.1.37) Malate dehydrogenase, L-malate:NAD⁺ oxidoreductase - (EC 1.18.1.3) NADH:ferredoxin reductase, ferredoxin:NAD⁺ oxidoreductase - (EC 3.1.4.1) Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase - (EC 2.3.1.8) Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase - (EC 2.3.1.9) Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase A preliminary account of this work has been given (Decker et al. 1976)     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris

Summary The addition of citrate to glucose broth led to an increase in specific growth rate and glucose catabolism, but a decrease in molar growth yield from glucose, in Leuconostoc mesenteroides subsp. cremoris. Acetate and formate were produced during the stationary phase of growth. According to the fermentation balance, part of the acetate and lactate came from the pyruvate of citrate metabolism. L. mesenteroides subsp. cremoris incorporated radioactive metabolites from [1,5-¹⁴C] citrate into cell material, primarily into lipids. [U-¹⁴C] Glucose was not incorporated into cell material.     

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.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

Effect of (--)-hydroxycitrate on ipomeamarone biosynthesis from pyruvate in sweet potato with black rot

(—)-Hydroxycitrate, a potent inhibitor of ATP: citrateoxaloacetate-lyase inhibited about 90% and 30% respectively,of the conversion of citrate-1,5^-14C and pyruvate-3^-14C intoipomeamarone, in sweer potato root tissuw infected with Ceratocystisfimbriota. However, the conversion of acetate-2¹⁴C into ipomeamaronewas not affected by (—)-hydroxycitrate. These resultssuggest that ATP: citrate oxaloacetate lyase plays a role inthe supply of acetyl CoA for terpenoid formation. ¹This paper constitutes part 109 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received July 29, 1973; )     

In vivo citrate studies with developing soybean cotyledons

The in vivo data presented here are strong evidence for theinvolvement of citrate cleavage enzyme in lipid synthesis indeveloping soybean cotyledons. The incorporation of ¹⁴C fromcitrate into crude lipid fraction in vivo had a pH optimum of4.5; was linear with time; had a temperature optimum of 35?C;and was inhibited by (–)-hydroxycitrate. The point ofcitrate cleavage was between carbons 3 and 4 of the citratemolecule and therefore ¹⁴C was incorporated into crude lipidfraction from citrate-5-¹⁴C but not citrate-1-¹⁴C or citrate-6-¹⁴C. ¹ Cooperative investigations of the Agricultural Research Service,U.S. Department of Agriculture, and the Illinois AgriculturalExperiment Station. ² This research represents partial fulfillment of the Ph.D.requirements of Daniel R. Nelson. Presently at Monsanto AgriculturalProducts Co., St. Louis, MO 63141, U.S.A. (Received September 20, 1976; )     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: II. Intermediary Metabolism and the Location of Control Points

Long term feeding of acetate-2-¹⁴C, ¹⁴CO₂, citrate-1,5-¹⁴C, fumarate-2,3-¹⁴C, and succinate-2,3-¹⁴C to mung bean (Phaseolus aureus L. var. Mungo) leaves in the dark gave labeling predominantly in tricarboxylic acid cycle intermediates. Kinetics of the intermediates during dark/light/dark transitions showed a light-induced interchange of ¹⁴C between malate and aspartate, usually resulting in an accumulation of ¹⁴C in malate and a decrease of it in aspartate. ¹⁴C-Phosphoenolpyruvate also showed a marked decrease during illumination. Changes in other intermediates of the tricarboxylic acid cycle were relatively minor. The kinetic data have been analyzed using the Chance crossover theorem to locate control points during the dark/light/dark transitions. The major apparent control points are located at malate and isocitrate dehydrogenases, and less frequently at citrate synthase and fumarase. These findings are explained in terms of the light-induced changes in adenine nucleotides and nicotinamide adenine dinucleotides.     

Kynurenines Impair Energy Metabolism in Rat Cerebral Cortex

Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of l-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington’s disease, Parkinson’s disease and Alzheimer’s disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study, we investigated the in vitro effect of l-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA) on some parameters of energy metabolism, namely glucose uptake, ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate, as well as on the activities of the respiratory chain complexes I–IV and Na⁺,K⁺-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except l-kynurenine, significantly increased glucose uptake and inhibited ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by 3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I–III activities. Moreover, Na⁺,K⁺-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine intermediates provoke impairment of brain energy metabolism.     

Propionate in heme biosynthesis in soybean nodules

When soybean nodules are incubated with propionate-2-¹⁴C the heme moiety of leghemoglobin becomes labeled. The incorporation of propionate-2-¹⁴C into heme is linear with time and it appears that propionate is utilized without a lag period. The rate of incorporation of propionate-2-¹⁴C into heme is more rapid than the rate of incorporation of succinate-2-¹⁴C and citrate-1,5-¹⁴C, however, these rates of incorporation may be influenced by different sizes of endogenous pools of organic acids.     

Oligomeric forms of bacterial malate dehydrogenase: a study of the enzyme from the phototrophic non-sulfur bacterium Rhodovulum steppense A-20s

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the phototrophic purple non-sulfur bacterium Rhodovulum steppense A-20s. According to gel-chromatography and electrophoretic studies, malate dehydrogenase is present as a dimer, tetramer and octamer depending on cultivation conditions. In phototrophic aerobic conditions only the tetrameric form was present, in chemotrophic aerobic conditions all three forms were detected, while in the absence of oxygen the octameric form disappeared. The malate dehydrogenase oligomers are encoded by a single gene and composed of the same 35 kDa polypeptide but differ in pH and temperature optimum, in affinities to malate, oxaloacetate, NADH and NAD⁺ and in regulation by cations and citrate. By modulating the cultivation conditions, it has been established that the dimer participates in the glyoxylate cycle; the tetramer operates in the tricarboxylic acid cycle, and the octamer may be involved in the adaptation to oxidative stress.