Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme.

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria-Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV-Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis-Menten mechanism (K (m) = 97 ± 11 µM, V = 9.4 ± 0.5 µM min(-1), and k (cat) = 12.1 ± 0.6 s(-1)) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

A simple micromethod for rapid kinetic analyses of yeast enzymes in situ.

A simple approach for rapid assay of enzymes in large numbers of samples of whole cells of yeast is described. The technique involves treatment of yeast cells with toluene dissolved in ethanol (1:4, v/v; TE), rendering them selectively permeable to small molecules, while letting the enzymes remain intracellular and catalytically active but accessible to exogenously added assay reagents. The permeated cell preparations permit rapid assay of nitrate reductase activity in situ [P. V. Choudary and G. Ramananda Rao (1976) Current Sci. 45, 324-327]. Investigation of enzyme kinetic parameters such as pH optima, temperature dependence, and substrate saturation, which involves several samples and needs to be investigated very rapidly and accurately, is greatly facilitated by permeated cells prepared from a very small culture of cells, eliminating the need for elaborate preparations of cell extracts. By using permeabilized cell preparations, the need to supplement the reaction mixture with expensive cofactors can also be obviated to a large extent. Further, the enzyme activity displays a broader range of tolerance in situ to variations in reaction conditions compared to that in vitro. Permeabilized cell preparations thus seem to present the enzymes in the form of relatively less disintegrated functional complexes, making them a valuable tool in biochemical and cell biological investigations, and facilitate the correlation of in vitro data with the in vivo phenomena more confidently.

Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulated Pisum sativum L.

Effect of nitrate on the nitrogenase (C2H2-reduction) activity, growth of nodule tissue accumulation of nitrate and nitrate reductase activity in 4-weeks-old nodulated peas (Pisum sativum L.) was investigated. A relatively slow decrease of the total nitrogenase activity (mumol C2H4 per root per h), as compared with plants cultivated without nitrate, was due to both retardation of further growth of the nodule tissue and to a decrease of their specific nitrogenase activity (mumol C2H4 per gf.wt. per h). However, an absolute and pronounced decrease of both nitrogenase activities occurred only 4 or 7 d after the application of nitrate. The addition of nitrate led to its rapid accumulation in the nodule and leaf tissue with a simultaneous induction of the nitrate reductase activity. The nitrogenase activity was not completely inhibited even after a 7-d cultivation with 280 m NO3- -N in the nutrient medium and after accumulation of up to 180 ppm NO3- -Nf.wt. in the nodule tissue. The results obtained indicate that the "photosynthate deprivation" reflects competition between assimilation of nitrate and fixation of dinitrogen.

In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants.

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.