Identification of Escherichia coli YgaF as an L-2-hydroxyglutarate oxidase

YgaF, a protein of previously unknown function in Escherichia coli, was shown to possess noncovalently bound flavin adenine dinucleotide and to exhibit L-2-hydroxyglutarate oxidase activity. The inability of anaerobic, reduced enzyme to reverse the reaction by reducing the product alpha-ketoglutaric acid is explained by the very high reduction potential (+19 mV) of the bound cofactor. The likely role of this enzyme in the cell is to recover alpha-ketoglutarate mistakenly reduced by other enzymes or formed during growth on propionate. On the basis of the identified function, we propose that this gene be renamed lhgO.     

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Two omega-amino acid transaminases from Bacillus cereus.

Bacillus cereus strain K-22 produced two distinct omega-amino acid transaminases, one catalyzing the transamination between beta-alanine and pyruvic acid and the other that between gamma-aminobutyric acid and alpha-ketoglutaric aic. The two enzymes were partially purified and separated from each other by various chromatographies. beta-Alanine:pyruvic acid transaminase and gamma-aminobutyric acid:alpha-ketoglutaric acid transaminase were induced by the addition of beta-alanine and gamma-aminobutyric acid, respectively, to the growth medium. beta-Alanine transaminase showed an optimum pH of 10.0 and optimum temperature of 35 degrees C, and its Km values for beta-alanine and pyruvic acid were both 1.1 mM. gamma-Aminobutyric acid, epsilon-aminocaproic acid, 2-aminoethylphosphonic acid, and propylamine showed about 30-40% of the activity of beta-alanine as amino donors, and oxalacetic acid was as good an amino acceptor as pyruvic acid. The optimum pH and temperature of gamma-aminobutyric acid transaminase were 9.0 and 50 degrees C, respectively, and its Km value for gamma-aminobutyric acid was 2.8 mM, while that for alpha-ketoglutaric acid was 2.3 mM. gamma-Aminobutyric acid and delta-aminovaleric acid were good amino donors but other omega-amino acids were virtually inactive with gamma-aminobutyric acid transaminase; alpha-ketoglutaric acid, and to a lesser extent glyoxylic acid, were active amino acceptors. Sulfhydryl reagents specifically activated gamma-aminobutyric acid transaminase.     

Characterization of an aromatic amino acid aminotransferase from Rhizobium leguminosarum biovar trifolii.

The most abundant aromatic amino acid aminotransferase of Rhizobium leguminosarum biovar trifolii was partially purified. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration. The enzyme transaminated aromatic amino acids and histidine. It used aromatic keto acids and alpha-ketoglutaric and oxalacetic acids as amino-group acceptors. The optimum temperature was 35 degrees C. Using phenylalanine and alpha-ketoglutaric acid as substrates the activation energy was 46.2 kJ.mol-1 and for the couple tryptophan:alpha-ketoglutaric acid it was 70.3 kJ.mol-1. The optimum pH was different for each substrate: 7.3 for phenylalanine, 7.9 for histidine and 8.7 for tryptophan.     

Mechanism of Proline Production by Kurthia catenaforma

The fate of aspartic acid used for proline fermentation by Kurthia catenaforma was traced by using aspartic acid-U-(14)C. The radioactivities of proline and glutamic acid increased with the disappearance of aspartic acid. After 40 hr, aspartic acid disappeared from the medium and radioactive alpha-ketoglutaric acid was detected. The radioactivity of proline reached 44% of aspartic acid radioactivity at 40 hr. The specific radioactivities of these amino acids and of alpha-ketoglutaric acid supported the notion that proline is produced mainly from aspartic acid via alpha-ketoglutaric acid and glutamic acid. Since the levels of glutamic acid dehydrogenases (EC 1.4.1.2 and EC 1.4.1.4) were low in this organism, it appears that the nitrogen atom of aspartic acid enters proline by the action of aspartate aminotransferase (EC 2.6.1.1). The mechanism of proline production is discussed on the basis of the role of aspartic acid in this fermentation.     

Growth of Azotobacter chroococcum strains on different substrates]

Azotobacter chroococcum 34, actively growing on alpha-ketoglutaric acid, and Azotobacter chroococcum B and KL, which almost do not assimilate this acid, can grow on the majority of substrates of the tricarboxylic acid cycle and on glucose, with and without nitrogen. The rate of assimilation of alpha-ketoglutaric acid is several times higher in the cells cultivated in the presence of this acid than in the cells grown on other substrates. This difference seems to involve the mechanism of transport of alpha-ketoglutaric acid into the cell.     

Effect of organic acids on the biosynthesis of carotenes by an Actinomyces chrysomallus strain

Synthesis of carotenes by Actinomyces chrysomallus var. carotenoides was stimulated by citric, acetic, oxalacetic, fumaric, succinic, malic, alpha-ketoglutaric, tartaric, pyruvic, and propionic acids. Acetic acid acts as a precursor of carotene synthesis and also has another stimulating mechanism of action on carotenogenesis of the actinomycete. Acetic, furmaric, malic, succinic, and alpha-ketoglutaric acids stimulate cyclization of lycopene yielding beta-carotene.     

Transamination of LTE4 by cysteine conjugate aminotransferase

Leukotriene E4 (LTE4) was transaminated to 5(S)-hydroxy-6(R)-S-(2-keto-3-thiopropionyl)-7,9-trans-11,14-cis- eicosatetraenoic acid (tentatively designated as LTG4) by cysteine conjugate aminotransferase I purified from rat liver supernatant in the presence of alpha-ketoglutaric acid or alpha-keto-gamma-methiolbutyric acid. The transamination activity was present in the kidney as well as in the liver, but not in the lung or leukocytes.     

Excretion of alpha-keto acids by strains of Streptomyces venezuelae

Cultures of Streptomyces venezuelae released acidic metabolites during nitrogen-limited growth on glucose. The main products were pyruvic acid and alpha-ketoglutaric acid. Variation in the extent of acid production was observed; spores of the parental strain 13s gave approximately 10% of low-producing colonies when plated on acid-base indicator medium. Examination of one low producer, strain PC 51-5, showed that differences in acid production became apparent only in low-glucose media containing manganese. In both strains PC 51-5 and 13s, uptake of alpha-keto-[5-14C]glutaric acid occurred by diffusion and no marked differences in permeability to alpha-ketoglutarate were detected. However, differences were observed in the activity of alpha-ketoglutarate dehydrogenase. In cultures of strain PC 51-5, the specific activity of the enzyme increased throughout growth, whereas in the parental strain activity decreased and could not be detected in older mycelium. Loss of enzyme activity was accompanied by excretion of alpha-ketoglutaric acid and failure to assimilate the product after glucose exhaustion. The results suggest that accumulation of pyruvic and alpha-ketoglutaric acids in S. venezuelae cultures grown in glucose-containing media may be due to regulatory suppression of the dehydrogenases by this carbon source.     

Chemical evolution of the citric acid cycle: Sunlight photolysis of α-ketoglutaric acid

Sunlight photolysis of alpha-ketoglutaric acid produces succinic acid as a major product. Other higher molecular weight products are identified by GC-MS analysis. These results provide further support for the important role of succinic acid in chemical evolution.     

Regulation of acetyl-CoA synthetase of Saccharomyces cerevisiae.

Acetyl-coenzyme A synthetase (EC 6.2.1.1) activity of Saccharomyces cerevisiae was determined by a radioactive assay procedure. The activity in vitro was inhibited significantly by NADPH, NADH, or AMP and to a lesser extent by NADP, NAD, or ADP. Glutamic acid and alpha-ketoglutaric acid were not inhibitory. The enzyme level was repressed when the cells were grown in a complex nutrient medium as opposed to the minimal medium. However, a glutamic acid auxotroph glul, when grown in excess glutamic acid, demonstrated a fivefold increase of acetyl-CoA synthetase.     

Preparation and properties of modified chitosan as potential matrix materials for drug sustained-release beads

Modified chitosan such as chitosan alpha-ketoglutaric acid (KCTS) and hydroxamated chitosan alpha-ketoglutaric acid (HKCTS) were successfully prepared. The modified chitosan were employed in the formation of drug-loaded, iron(III)-crosslinked polymeric beads. The produced polymers were characterized by IR, NMR, WXRD and DSC measurements. The resulting beads were evaluated in vitro as drug prolonging and potentially orally administered delivery system. Theophylline was used as the loaded model drug. The generated beads proved to be successful in prolonging drug release. The release kinetics was evaluated by fitting the experimental data to standard release equations (zero-, first- and Higuchi equation). The best fit was found with Higuchi model for the polymeric beads.     

Inhibition of rat liver rhodanese by di-, tricarboxylic, and alpha-keto acids.

Rat liver rhodanese [EC 2.8.1.1] purified by ammonium sulfate fractionation, CM-cellulose and Sephadex G-200 chromatography yielded two active fractions (I & II). Their molecular weights were estimated to be 1.75 X 10(4) (I) and 1.26 X 10(4) (II) by the gel filtration method. Kinetic studies revealed that Fraction I rat liver rhodanese catalyzes thiocyanate formation from thiosulfate and cyanide by a double displacement mechanism. Carboxylic acids such as DL-isocitric, citric malic, pyruvic, and oxaloacetic acid were competitive inhibitors with respect to thiosulfate, whereas fumaric, succinic, and alpha-ketoglutaric acids were noncompetitive inhibitors with respect ot thiosulfate. Incubation of mitochondria with sulfate and alpha-ketoglutaric acid caused a significant decrease in rhodanese activity.     

The influence of rabies immunization of gamma-aminobutyric acid metabolism in the brains of animals]

Subcutaneous injection to albino rats (100-120 g) of lived fixed rabies virus was accompanied by a brief marked decrease in the content of gamma-aminobutryic acid in the animals' brain. There was also an increase in the activity of gamma-aminobutyric acid alpha-ketoglutaric acid transaminase in the brain tissue of animals vaccinated with live fixed rabies virus.     

D-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens

Cell-free extract (crude extract) of Agrobacterium tumefaciens grown on d-glucuronate or d-glucarate converts d-glucarate and galactarate to a mixture of 2-keto-3-deoxy- and 4-deoxy-5-keto-d-glucarate. These compounds are then converted by partially purified crude extract to an intermediate tentatively identified as 2,5-diketoadipate. The same enzyme preparation further decarboxylates this intermediate to alpha-ketoglutarate semialdehyde, which is subsequently oxidized in a nicotinamide adenine dinucleotide-dependent reaction to alpha-ketoglutaric acid. Since A. tumefaciens converts d-glucuronic acid to d-glucarate, a pathway from d-glucuronate to alpha-ketoglutarate in A. tumefaciens was determined.     

Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and alpha-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4, 6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F(420), and methanofuran, respectively. Both the MJ1425-encoded enzyme and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant. Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V(max)/K(m) value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nucleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F(420). Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.     

Biochemical characterization of the yeast Yarrowia lipolytica overproducing carboxylic acids from ethanol. Nitrogen metabolism enzymes

A comparative assay of nitrogen metabolism enzymes in the Yarrowia lipolytica mutant N1 grown under conditions promoting the overproduction of either alpha-ketoglutaric acid (KGA) or citric acid showed that the overproduction of KGA correlates with an increase in the activities of the NAD- and NADP-linked glutamate dehydrogenase, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase reactions. These reactions are likely to be responsible for the overproduction of KGA by this mutant. In contrast, the overproduction of citric acid correlated with a decline in the activities of the NAD- and NADP-linked glutamate dehydrogenases and with an increase in the activities of glutamine synthetase and glutamate synthase.     

Catabolism of amino acids in bacteria of the genus Klebsiella

Propagation and activity level of 18 enzymes catalyzing deamination reactions of dicarboxylic and oxyamino acids and enzymes of amino acid reamination and amino acid N-acyl-derivatives' deacylation have been studied in Klebsiella bacteria. Klebsiella the most actively utilizes serin, threonine, aspartic and glutamic acids and aromatic amino acids. The first three amino acids are utilized by deamination, aromatic acids- in aminotransferase reaction with alpha-ketoglutaric acid, glutamic acid--by deamination and decarboxylation. Besides, Klebsiella actively deacylates N-acyl-derivatives of amino acids.     

Production of organic acids by bacteria isolated from soil, rhizosphere and mycorrhizosphere of pine (Pinus sylvestris L.)

The bacteria studied released into the medium ten to eleven organic acids. Soil organisms excreted mainly pyruvic and alpha-ketoglutaric acids, while strains from the root zone--gluconic acid and unidentified uronic acid (y2). Mean indices of total production of the organic acids by bacteria were in the following order: rhizosphere less than soil less than mycorrizosphere. Bacteria from the root zone released into the medium very large amounts of pyruvic, gluconic, and uronic (y2) acids--in some instances several times higher than bacterial dry mass.     

Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of alpha-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes

A comparative study of the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing alpha-ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.