Loss Of Nitrate Reductase Activity Research Articles

Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O(2)), while no visible colonies were formed in the absence of O(2). However, in the presence of nitrate (NO3-), the organism exhibited limited growth anaerobically with production of nitrite (NO2-), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using L-lysine- and L-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha

Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.

Nitrate reductase inactivator from Spirodela polyrhiza (L.) Schleiden

NADH:nitrate reductase (EC 1.6.6.1) activity in the crude extract from Spirodela polyrhiza was relatively labile in vitro. Inclusion of polyvinylpolypyrrolidone into the extraction medium had only a slight effect on the stability of the enzyme, whereas addition of 3 % casein, azocasein, or other proteins to the extraction medium greatly increased the nitrate reductase (NR) activity. Various protease inhibitors were tested for their ability to prevent the loss of NR activity in vitro. Iodoacetate and para-chloromercuric benzoate, the thiol-protease inhibitors, as well as pepstatin, the aspartic-protease inhibitor had no effect on stability of the nitrate reductase. EDTA had a slight stimulatory effect, whereas 5 mM o-phenantroline, another inhibitor of the metallo-proteases increased the activity of nitrate reductase. The highest enzyme activity was found in the presence of phenylmethylsulphonyl fluoride and di-isopropyl phosphorofluoridate both being serine-protease inhibitors.

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of Light).

Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d-old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a light/dark regime for 5 d and then induced with 5 mM KNO3 for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabolites rather than to immediate products of the light reactions of photosynthesis.

Biochemical genetics of further chlorate resistant, molybdenum cofactor defective, conditional-lethal mutants of barley

Three plants, R9201 and R11301 (from cv. Maris Mink) and R12202 (from cv. Golden Promise), were selected by screening M2 populations of barley (Hordeum vulgare L.) seedlings (mutagenised with azide in the M1) for resistance to 10 mM potassium chlorate. Selections R9201 and R11301 were crossed with the wild-type cv. Maris Mink and analysis of the F2 progeny showed that one quarter lacked shoot nitrate reductase activity. These F2 plants also withered and died in the continuous presence of nitrate as sole nitrogen source. Loss of nitrate reductase activity and withering and death were due in each case to a recessive mutation in a single nuclear gene. All F1 progeny derived from selfing selection R12202 lacked shoot nitrate reductase activity and also withered and subsequently died when maintained in the continuous presence of nitrate as sole nitrogen source. All homozygous mutant plants lacked not only shoot nitrate reductase activity but also shoot xanthine dehydrogenase activity. The plants took up nitrate, and possessed wild-type or higher levels of shoot nitrite reductase activity and NADH-cytochrome c reductase activity when treated with nitrate for 18 h. We conclude that loss of shoot nitrate reductase activity, xanthine dehydrogenase activity and withering and death, in the three mutants R9201, R11301 and R12202 is due to a mutation affecting the formation of a functional molybdenum cofactor. The mutants possessed wild-type levels of molybdenum and growth in the presence of unphysiologically high levels of molybdate did not restore shoot nitrate reductase or xanthine dehydrogenase activity. The shoot molybdenum cofactor of R9201 and of R12202 is unable to reconstitute NADPH nitrate reductase activity from extracts of the Neurospora crassa nit-1 mutant and dimerise the nitrate reductase subunits present in the respective barley mutant. The shoot molybdenum cofactor of R11301 is able to effect dimerisation of the R11301 nitrate reductase subunits and can reconstitute NADPH-nitrate reductase activity up to 40% of the wild-type molybdenum cofactor levels. The molybdenum cofactor of the roots of R9201 and R11301 is also defective. Genetic analysis demonstrated that R9201, but not R11301, is allelic to R9401 and Az34 (nar-2a), two mutants previously shown to be defective in synthesis of molybdenum cofactor. The mutations in R9401 and R9201 gave partial complementation of the nar-2a gene such that heterozygotes had higher levels of extractable nitrate reductase activity than the homozygous mutants.

Comparative biochemical characterization of mutants at the nitrate reductase/molybdenum cofactor loci cnxA, cnxB, and cnxC of Nicotiana plumbaginifolia

Mutations in any of the three gene loci cnxA, cnxB, cnxC can lead to a total loss of nitrate reductase activity in Nicotiana species. The cnx loci are involved in synthesis and processing of the molybdenum cofactor, which is an essential structural constituent of nitrate reductase. The biochemical properties of cnxA, cnxB and cnxC mutant cell lines of Nicotiana plumbaginifolia were examined further. The cnxA line (N×9) was found to possess a catalytically defective but dimerization-active and under in vivo/in vitro-conditions repairable molybdenum cofactor, thus, resembling the properties of N. tabacum cnxA lines. The cnxB (N×24) and cnxC (N×21) mutants. however, show a phenotype very different from cnxA. This new phenotype is characterised by an irreversible loss of both the catalytic function and dimerization ability of the molybdenum cofactor which makes it likely that the molybdopterin moiety of the cofactor is defective or lacking in these mutants. In this report we summarize and compare the phenotypic data presently available for the Nicotiana loci cnxA, cnxB and cnxC. Possible functions of the gene products of these loci will be discussed.

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato (Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.

Differential Senescence of Maize Hybrids following Ear Removal

In conjunction with a study of the effects of ear removal on the senescence of whole maize (Zea mays L.) plants, visual symptoms and associated changes in constituent contents and activities of a selected leaf (first leaf above the ear) were determined. Leaves were sampled from field-grown eared and earless Pioneer brand 3382, B73 x Mo17, and Farm Services brand 854 maize hybrids at nine times during the grainfilling period.VISUAL SYMPTOMS INDICATED THE FOLLOWING SEQUENCE AND RATE OF SENESCENCE: earless B73 x Mo17 > earless P3382 >> eared B73 x Mo17 >> eared P3382 </= earless FS854 > eared FS854. All earless hybrids showed increases in leaf dry weight and sugar content; however, the increases were transitory for P3382 and B73 x Mo17, but continuous throughout the grain-filling period for FS854, indicative of continued photosynthetic activity of the latter. All earless hybrids exhibited similar and transitory starch accumulation patterns. Thus, FS854 was an exception to the concept that carbohydrate accumulation accelerates leaf senescence. Ear removal resulted in accelerated losses of reduced N, phosphoenolpyruvate and ribulose bisphosphate carboxylases, phosphorus, chlorophyll, nitrate reductase activity, and moisture for P3382 and B73 x Mo17 plants. In contrast, the loss of all components (except phosphorus) was similar for the selected leaf of earless and eared FS854.Although the loss of nitrate reductase activity, reduced N, and carboxylating enzymes accurately reflected the development of senescence of the selected leaf, the rate of net loss of reduced N and carboxylating enzymes appeared to be regulated. We deduced that the rate of flux of N into the leaf was a factor in regulating the differing rates of senescence observed for the six treatments; however, we cannot rule out the possibility of concurrent influence of growth regulators or other metabolites.

A conditional-lethal cnx-type nitrate reductase-deficient barley mutant

Selection for resistance to chlorate in an M2 population of barley (Hordeum vulgare cv. Maris Mink, M1 seeds treated with sodium azide) yielded nine green plants with zero or very low in vivo nitrate reductase activity. One of these plants, R9401, was crossed with the wild-type cv. Golden Promise and analysis of the F2 progeny showed that both loss of nitrate reductase activity and resistance to chlorate was due to a recessive mutation in a single nuclear gene. Homozygous mutant plants did not grow on nitrate as sole nitrogen source, in vermiculite or in sterile culture. They grew aseptically on ammonium nitrate, ammonium citrate or urea, although glutamine was the best nitrogen source. Homozygous mutant plants possessd <1% of wild-type in vitro NADH-nitrate reductase activity and lacked detectable xanthine dehydrogenase activity. The plants possessed normal nitrite reductase activity and an inducible NADH-cytochrome c reductase activity. We conclude that loss of nitrate reductase activity is due to a cnx-type mutation affecting the formation of a functional cofactor.

Effect of salicylic acid on nitrate reductase activity in maize seedlings

The effect of different concentrations of salicylic acid on total Kjeldahl nitrogen and nitrate reductase activity in the maize (Zea mays L.) seedling was studied. The total nitrogen of the maize embryonic axis (root + shoot) from seedlings raised with 10 mM Ca(NO3)2 for 5 days was substantially higher than that from the control when 0.01 mM salicylic acid was supplied. As supply of high (1 mM) concentrations of salicylic acid decreased the accumulation of organic nitrogen. The in vivo activity of nitrate reductase in the roots increased at low concentrations of salicylic acid, while high concentrations were inhibitory. The stimulative concentration of the acid protected in vivo loss of nitrate reductase activity under non‐inducing conditions, whereas it had no effect on in vitro loss of enzyme. It is suggested that salicylic acid increases in vivo enzyme activity indirectly, to some extent by protecting the natural inactivation of the enzyme.

Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa.

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.

Some New Aspects of the in Vivo Assay for Nitrate Reductase in Wheat (Triticum aestivum L.) Leaves

Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millimolar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed.

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 mum canavanine was effective in root tips whereas only 50 mum was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO(3) resulted in a loss of NRA. NO(3) (-) protected nitrate reductase from this inactivation in both root tip and mature root sections.

Regulation of the Neurospora crassa assimilatory nitrate reductase.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source.

Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves

A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH(2)-nitrate reductase and NO(3) (-)-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A x 182E). The three activities were induced in parallel in both tissues when NO(3) (-) was supplied. WO(4) (=) suppressed the induction of NADH- and FMNH(2)-nitrate reductase activities in root tips and leaves. The NO(3) (-)-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO(4) (=). In leaves, on the other hand, there was a marked superinduction of the NO(3) (-)-induced NADH-cytochrome c reductase in the presence of WO(4) (=).The half-life values of NADH-nitrate reductase and FMNH(2)-nitrate reductase measured by removing NO(3) (-) and adding WO(4) (=) to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO(3) (-) in the induction medium together with WO(4) (=) gave partial protection of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities in both root tips and leaves with a t(0.5) of 6 and 8 hours, respectively. NO(3) (-) also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO(3) (-)-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO(3) (-) protected the loss of NO(3) (-)-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase.

The Regulation of Nitrate Reductase in the Fungus Aspergillus nidulans

Nitrate reductase induction by nitrate and repression by NH,+ has been studied extensively in the simple eukaryote Aspergillus nidulans (Pateman & Cove, 1967; Cove & Pateman, 1968). Wild-type cells grown on minimal medium, with 20m~-NaNO~ as the nitrogen source, rapidly lose nitrate reductase activity when transferred either to minimal medium without N (-N medium) or to minimal medium without C and N (-CN medium) or to -CN medium plus 100m-L-glutamate as the sole N and C source. Hynes (1973), whose work we have confirmed, found that transferring pre-grown cells from 10mM-NaN03 to a medium lacking a C source (-C medium) resulted in the greatest decrease in activity, even in the presence of 10m-NaN03. Cyclohexamide, an inhibitor of protein synthesis at a concentration of lOpg/ml, did not prevent the loss of activity. When cell-free extracts of wild-type cells, grown on -N medium+20m~-NaNO~, were centrifuged at 6OOOOg for 20min and incubated for periods of up to 1 h with 2.65m1 of orthophosphate buffer, pH7.75,+3.5pg of FAD, there was a rapid loss of nitrate reductase activity (see Fig. 1). There was a similar loss of enzyme activity when either 1lOm-NaNO3 or 35m-NH was,+ added to the incubation mixture. When NADH-linked nitrate reductase extracts from Nitrobacter agilis, pre-grown on nitrate, are incubated in the absence of nitrate, rapid inactivation of the enzyme results. Herrera & Nicholas (1974) found that this inactivation was the result of over-reduction with NADH. Several workers also found that nitrate reductase is inactivated by reduced

SODIUM RELATIONS IN DESERT PLANTS

Atriplex confertifolia (Torr, Frem.) Wats. seedlings were grown in soil irrigated with different salt levels. Maximum yields were obtained with a 50 me/liter NaCl treatment. Higher salt levels produced large, succulent leaves showing symptoms of nitrogen deficiency. Physiological effects of salt were noted as decreased chlorophyll, soluble protein, and nitrate and loss of nitrate reductase activity. Salt also reduced the uptake of Mg, K, and P. Net carbon assimilation rates were reduced presumably from salt-induced increases in respiration. Leaf water potential was decreased greatly by the salt levels, but transpiration levels at leaf temperature of 20°C did not correspond in that they were quite insensitive to salt levels. At 30°C, transpiration rates decreased with increasing salt indicating an improved water use efficiency.

Reduced nicotinamide-adenine dinucleotide-nitrite reductase from Azotobacter chroococcum.

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.

DISAPPEARANCE OF NITRATE REDUCTASE ACTIVITY FROM CHLAMYDOMONAS REINHARDI

Nitrate reductase activity was induced in Chlamydomonas reinhardi following addition of nitrate. Enzymic activity was assayed in cell‐free supernatants and in whole cells whose permeability had been increased by freezing. Nitrate reductase activity in cells decreased rapidly when CO2‐fixation was prevented by (a) darkening cultures, (b) aerating cultures with CO2‐free air, or (c) addition of DCMU (3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea). A smaller loss of nitrate reductase activity from darkened cells occurred when (a) acetate‐adapted cells were supplied with acetate, or (b) cells were allowed to accumulate carbon reserves by nitrogen starvation before darkening. It was concluded that maintenance of nitrate reductase activity was dependent upon the availability of a suitable carbon source.