Characterization of molybdenum-free nitrate reductase from haloalkalophilic bacterium Halomonas sp. strain AGJ 1-3.

Nitrate reductase from the haloalkalophilic denitrifying bacterium Halomonas sp. strain AGJ 1-3 was isolated and purified to homogeneity. The isolated enzyme belongs to a novel family of molybdenum-free nitrate reductases. It presents as a 130-140 kD monomeric protein with specific activity of 250 micromol/min per mg protein. The enzyme reduces not only nitrate, but also other anions, thus showing polyoxoanion reductase activity. Enzyme activity was maximal at pH 7.0 and 70-80 degrees C.

Isolation, purification, and characterization of nitrate reductase from a salt-tolerant Rhodotorula glutinis yeast strain grown in the presence of tungsten.

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.

[Properties of nitrate reductase from Fusarium oxysporum 11dn1 fungi grown under aerobic and anaerobic conditions].

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.

[Role of trophic and hormonal factors in exogenous regulation of the formation of reproductive organs in yellow lupine (Lupinus luteus L.)]. / Rol' troficheskogo i gormonal'nogo factorov v ékzogennoi reguliatsii formirovaniia reproduktivnykh organov u liupina zheltogo (Lupinus luteus L.).

During the formation and ripening of beans in yellow lupine, their intactness may be increased by treatment of the plants with the microelement molybdenum or the growth factor 24-epibrassinolide. Early stages of the effects of either factor involved activation of various metabolic pathways; eventually, the supply of fruit elements with free amino acids was improved and the seed productivity of the plants, increased.

[Reduction of nitrates by cultures of Azotobacter indicum and Azotobacter chroococcum ]. / Vosstanovlenie nitratov kul'turami Azotobacter indicum i Azotobacter chroococcum.

The capacity for denitrification was studied in Azotobacter bacteria, which are free-living nitrogen-fixing obligatory aerobes. Data on the nitrate reduction to nitrites and nitric oxide by A. indicum under anaerobic conditions were obtained for the first time for genus Azotobacter.

Tungsten-containing enzymes.

The biological importance of tungsten has been fully proved in the last decade due to isolation of a number of tungsten-containing enzymes (W-enzymes) from hyperthermophilic archaea. Tungsten was previously considered only as an antagonist of molybdenum, because the replacement of molybdenum by tungsten (due to their chemical similarity) leads to inactivation of molybdenum-containing enzymes (Mo-enzymes). In addition to the "true W-enzymes" in which tungsten cannot be replaced by molybdenum, recently some enzymes have been isolated which can use either molybdenum or tungsten in the catalytic process. This review briefly summarizes data on the participation of tungsten in catalysis by some enzymes and the structure of the active sites of W-enzymes.

[Kinetic method of tungsten analysis in tungsten-containing enzymes]. / Kineticheskii metod opredeleniia vol'frama v vol'fram-soderzhashchikh fermentakh.

A highly sensitive method for tungsten detection in proteins based on the ability of this metal to catalyze the oxidation of with hydrogen peroxide is described. The method allows determining tungsten in protein samples in the concentration range of -0.05 to -0.4 microgram/ml. Molybdenum, at a concentration lower than half the concentration of tungsten, as well as iron, selenium, and pterin at concentrations -2.5 times higher than that of tungsten, had no effect on tungsten determination by this method.

Vanadate reduction by molybdenum-free dissimilatory nitrate reductases from vanadate-reducing bacteria.

Molybdenum- and molybdenum cofactor-free nitrate reductases recently isolated by us from vanadate-reducing bacteria Pseudomonas isachenkovii are likely to mediate vanadate reduction. During anaerobic growth of P. isachenkovii on medium supplemented with nitrate and vanadate, vanadate dissimilation was followed by nitrate consumption, and this process was associated with some structural reorganizations of nitrate reductases. The homogeneous membrane-bound nitrate reductase of P. isachenkovii reduced vanadate with NADH as an electron donor.

Nitrate reductase and nitrous oxide production by Fusarium oxysporum 11dn1 under aerobic and anaerobic conditions.

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively.

Vanadium-binding protein excreted by vanadate-reducing bacteria.

A vanadium-binding protein was isolated from the culture medium of the vanadium-reducing bacterium Pseudomonas isachenkovii by utilizing vanadate as the terminal electron acceptor upon anaerobic respiration. The protein was associated with vanadium at a molar ratio of approximately 1:20. It was purified to homogeneity and separated into three components by treatment with 1 M HCl followed by gel filtration: a protein, a vanadium-binding ligand, and inorganic vanadium. Electron paramagnetic resonance analysis showed that vanadium was associated with the protein in the 4+ oxidation state. The distribution of vanadium within the cell was studied by electron microscopy and x-ray microanalysis of P. isachenkovii cells. The results suggest that vanadium, accumulated in special swells on the surface of the cell membranes, is reduced and excreted to the medium.

Stimulation of nitrate reductase activity of the salt-tolerant yeast Rhodotorula glutinis by tungsten in the presence of molybdenum.

Tungsten in the presence of molybdenum stimulates nitrate reductase activity and growth of the salt-tolerant yeast Rhodotorula glutinis on medium with nitrates. Tungsten is not incorporated in proteins possessing nitrate reductase activity. A significant increase in molybdenum cofactor in cells grown on medium with equimolar amounts of molybdenum and tungsten may relate to the stimulatory action of tungsten.

Molybdenum-free nitrate reductases from vanadate-reducing bacteria.

Two catalytically distinct molybdenum-free dissimilatory nitrate reductases, a soluble periplasmic and a membrane-bound one, were isolated from the vanadate-reducing facultatively anaerobic bacterium Pseudomonas isachenkovii and purified to electrophoretic homogeneity. The enzymes did not contain molybdenum, the periplasmic enzyme contained vanadium, whereas the membrane-bound enzyme was vanadium-free. Both nitrate reductases lacked molybdenum cofactor. This fact was proved by reconstitution of the apoprotein of the nitrate reductase of Neurospora crassa nit-1 mutant. This is the first demonstration of molybdenum-free and molybdenum cofactor-free nitrate reductases.

[Proteins of the lupin family, binding molybdenum, tungsten, and radionuclide effluents from the Chernobyl Atomic Energy Station]. / Belki semian liupina, sviazyvaiushchie molibden, vol'fram i radionuklidy vybrosov Chernobyl'skoi AES.

Yellow lupine seeds were found to contain two proteins and a low molecular weight fraction possessing the ability to bind Mo, W and radionuclides from the Chernobyl nuclear power in vivo and the 185W isotope in vitro. These proteins differ in their electrophoretic mobility. The electrophoretically less mobile protein undergoes proteolytic degradation; the proteolytic product retains the ability to accumulate microelements.

The nitrate reductase activity of milk xanthine oxidase.

Milk xanthine oxidase oxidizes xanthine at pH 9.6 and reduces nitrates at pH 5.2. It is shown that the nitrate reductase activity requires molybdenum and sulfur-containing sites in the enzyme, whereas oxidation of xanthine also requires iron-containing sites and FAD. As the pH changes from 5.2 to 9.6, the conformation of the enzyme molecule is modified as demonstrated by changes in the absorption, fluorescence, and circular dichroism spectra. When the enzyme is treated with dithioerythritol, it may pass from the oxidase to the dehydrogenase form with a marked increase in the nitrate reductase activity.

[Functional groups involved in the nitrate reductase activity of milk xanthine oxidase]. / Issledovanie funktsional'nykh grupp, uchastvuiushchikh v nitratreduktaznoi aktivnosti ksantinoksidazy moloka.

Milk xanthine oxidase possesses the nitrate reductase activity at pH 5.2; the pH optimum of the xanthine oxidase activity for the enzyme lies at 9.6. After removal of FAD and binding of Mo and Fe with a simultaneous measurement at the pH optima of the above activities it was found that only the Mo-containing site is necessary for the nitrate reductase activity. The switch-over of the enzyme from the xanthine oxidase to the nitrate reductase activity is associated with considerable conformational changes of the enzyme molecule.

[Photoinduction of carotenoid synthesis by Neurospora crassa on different structural and functional states of nitrate reductase]. / Fotoinduktsiia sinteza karotinoidov u Neurospora crassa pri raznom strukturnom i funktsinal'nom sostoianii nitratreduktazy.

In order to clarify the role of nitrate reductase as a potential photoreceptor, the ability of N. crassa mycelial cells for light-dependent accumulation of carotenoid pigments has been studied. The repression of the nitrate reductase synthesis by ammonium ions has been found not to influence the rate of the photoinduced carotenogenesis. The mutant experiments have shown that damage to the structural integrity of the nitrate reductase molecule, e, g., disintegrated synthesis of the protein fragment of the enzyme molecule (mutants nit-2 and nit-3) or the molybdenum coenzyme (mutant nit-1) does not affect the activity of the photoregulatory system of N. crassa. Thus, nitrate reductase is not a necessary component of the photoregulatory mechanism of N. crassa responsible for the synthesis of carotenoids.

[Nitrate and nitrite reductase activity of milk xanthine oxidase]. / Nitrat-i nitritreduktaznaia aktivnosti ksantinoksidazy moloka.

The nitrate and nitrite reductase activities of milk xanthine oxidases were studied. The optimal conditions for manifestation of these activities were found. A possible mechanism of electron transport to nitrate and nitrite inside the enzyme is discussed.

Isolation of cofactor common to molybdenum-containing enzymes: nitrate reductase from lupine bacteroids and xanthine oxidase from milk.

A quantitative method for the anaerobic isolation of a molybdenum cofactor from two molybdenum-containing enzymes, nitrate reductase from the bacteroids of lupine nodules and xanthine oxidase from milk, is described. It was established that the cofactor consists of an aromatic component and a number of amino acid residues bound to it. The structural and catalytic function of the molybdenum cofactor in the enzyme was established.