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PubMed2005

Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site.

Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

Animals↗
PubMed1979

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation.

All species of Rhizobium except R. lupini had nitrate reductase activity. Only R. lupini was incapable of growth with nitrate as the sole source of nitrogen. However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium. Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rhizobium trifolii, R. phaseoli, and R. leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate. In bacteroids of R. japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. trifolii, R. leguminosarum, and R. phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.

Culture Media↗
PubMed1994

Nitrate in baby foods. Adding to the nitrate mosaic.

OBJECTIVE: To identify commercial baby food varieties high in nitrate content using ion chromatography and compare the health risk associated with the consumption of high-nitrate water and high-nitrate commercial baby food. DESIGN: Ion chromatographic determination of nitrate concentration in a variety of commercial baby foods. SETTING: University Hygienic Laboratory, University of Iowa College of Medicine, Iowa City. PATIENTS: None. RESULTS: Commercial baby foods with nitrate levels higher than 45 ppm include mixed vegetables, bananas, carrots, garden vegetables, spinach, green beans, and beets. The amount of nitrate in one 113-g (4 oz) jar of beets, for example, is equivalent to the amount of nitrate in nearly 5.5 L of water at 45 ppm nitrate. CONCLUSIONS: A controlled clinical trial is needed to clarify how consuming high-nitrate foods correlates with methemoglobin levels in infants younger than 6 months.

Age Factors↗
PubMed1996

Does increased nitrate ingestion elevate nitrate levels in human milk?

OBJECTIVE: To determine whether the nitrate content of human milk is influenced by maternal ingestion of water containing elevated nitrate levels. DESIGN: Prospective, nonrandomized, volunteer study. SETTING: Clinical Research Center at the University of Iowa Hospitals and Clinics, Iowa City. PATIENTS: Twenty healthy lactating women with infants older than 6 months. INTERVENTIONS: The mothers were asked to consume a minimum of 1500 mL of water containing 0 mg of nitrate per liter on day 1, 45 mg on day 2, and 100 mg on day 3 in addition to consuming and recording their dietary intake. Breast-feeding was permitted during days 1 and 2, but milk was expressed on day 3 and the infants were given alternate food sources. After each 24-hour study day, maternal urine and milk samples were collected and frozen. A modified cadmium column reduction method was used to determine spot urinary and milk nitrate content. RESULTS: The meant total nitrate intake from diet and water on days 1,2, and 3, respectively, was 46.6, 168.1, and 272.0 mg. Spot urine nitrate content on days 1, 2, and 3, respectively, was 36.0, 66.0, and 84.0 mg. Nitrate concentration of human milk on days 1,2, and 3, respectively, was 4.4, 5.1 and 5.2 mg/L. CONCLUSION: Women who consume water with a nitrate concentration of 100 mg/L or less do not produce milk with elevated nitrate levels.

Adult↗
PubMed2003

The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans.

Dietary nitrate is metabolized to nitrite by bacterial flora on the posterior surface of the tongue leading to increased salivary nitrite concentrations. In the acidic environment of the stomach, nitrite forms nitrous acid, a potent nitrating/nitrosating agent. The aim of this study was to examine the pharmacokinetics of dietary nitrate in relation to the formation of salivary, plasma, and urinary nitrite and nitrate in healthy subjects. A secondary aim was to determine whether dietary nitrate increases the formation of protein-bound 3-nitrotyrosine in plasma, and if dietary nitrate improves platelet function. The pharmacokinetic profile of urinary nitrate excretion indicates total clearance of consumed nitrate in a 24 h period. While urinary, salivary, and plasma nitrate concentrations increased between 4- and 7-fold, a significant increase in nitrite was only detected in saliva (7-fold). High dietary nitrate consumption does not cause a significant acute change in plasma concentrations of 3-nitrotyrosine or in platelet function.

Adult↗
PubMed2007

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2-/- mice.

BACKGROUND AND PURPOSE: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. EXPERIMENTAL APPROACH: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. KEY RESULTS: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. CONCLUSIONS AND IMPLICATIONS: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways.

Aldehyde Dehydrogenase↗
PubMed1999

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.

Animals↗
PubMed1975

The influence of dietary nitrate on nitrate concentration in egg yolk and albumen.

Sodium nitrate (from 0 to 2,000 p.p.m.) was added to the drinking water of 32-week old S.C. White Leghorn pullets. Eggs collected once a week for 8 weeks were analyzed for nitrate content. Increasing levels of nitrate in the drinking water resulted in increasing levels of nitrate in albumen and yolk. Higher levels of nitrate were found in the yolk than in the albumen. When birds were started on the experiment, nitrate in the drinking water was reflected by immediate increases in nitrate content of the eggs. The nitrate content of eggs from birds receiving 1,000 p.p.m. NaNO3 (728 p.p.m. NO3-) exceeded the 45 p.p.m. permissible level of nitrate in drinking water for human beings.

Administration, Oral↗
PubMed1996

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots.

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 nM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.

Cell Line↗
PubMed2001

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

Cell Division↗
PubMed1999

Roles of nitrate, nitrite and ammonium ion in phytochrome regulation of nitrate reductase gene expression in maize.

The influence of nitrate and its metabolites on the nitrate reductase (NR) gene expression and its relationship with phytochrome (Pfr) regulation of NR in etiolated maize leaves is examined. Nitrate induction and Pfr stimulation are brought about by independent signalling phenomena. Phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), mimicked the effect of red light but could not replace the nitrate requirement for the induction of NR transcript accumulation. This suggests that while PKC-type enzymes may be involved in mediating the Pfr signal, nitrate may follow an independent signalling mechanism. Experiments with 5-hydroxytryptamine (5HT) and lithium ions (Li+), which are known to modulate phosphoinositide (PI) turnover, indicated that in addition to generating Pfr-induced second messengers for PKC activation, PI cycle may also generate other signals which mediate nitrate induction of NR gene expression in the dark. The products of nitrate reduction i.e., nitrite and ammonium ion had inhibitory and stimulatory effects respectively, on NR transcript accumulation. They work mainly at the level of nitrate induction.

Gene Expression Regulation, Enzymologic↗
PubMed2002

Elevated pCO(2 )favours nitrate reduction in the roots of wild-type tobacco (Nicotiana tabacum cv. Gat.) and significantly alters N-metabolism in transformants lacking functional nitrate reductase in the roots.

The impact of elevated pCO(2 )on N-metabolism of hydroponically grown wild-type and transformed tobacco plants lacking root nitrate reduction was studied in order to elucidate the effects on (i) nitrate uptake, (ii) long-distance transport of N, (iii) nitrate reduction with emphasis on root-NR, and (iv) the allocation of N between the root and shoot. The findings were related to alterations of growth rates. At elevated pCO(2 )the wild type exhibited higher growth rates, which were accompanied by an increase of NO(3)(-)-uptake per plant, due to a higher root:shoot ratio. Furthermore, elevated pCO(2 )enhanced nitrate reduction in the roots of the wild type, resulting in enhanced xylem-loading of organic N (amino-N) to supply the shoot with sufficient nitrogen, and decreased phloem-transport of organic N in a basipetal direction. Transformed tobacco plants lacking root nitrate reduction were smaller than the wild type and exhibited lower growth rates. Nitrate uptake per plant was decreased in transformed plants as a consequence of an impeded root growth and, thus, a significantly decreased root:shoot ratio. Surprisingly, transformed plants showed an altered allocation of amino-N between the root and the shoot, with an increase of amino-N in the root and a substantial decrease of amino-N in the shoot. In transformed plants, xylem-loading of nitrate was increased and the roots were supplied with organic N via phloem transport. Elevated pCO(2 )increased shoot-NR, but only slightly affected the growth rates of transformed plants, whereas carbohydrates accumulated at elevated pCO(2 )as indicated by a significant increase of the C/N ratio in the leaves of transformed plants. Unexpectedly, the C/N balance and the functional equilibrium between root and shoot growth was disturbed dramatically by the loss of nitrate reduction in the root.

Carbon Dioxide↗
PubMed2006

Glutamine transport and feedback regulation of nitrate reductase activity in barley roots leads to changes in cytosolic nitrate pools.

The size of tissue amino acid pools in plants may indicate nitrogen status and provide a signal that can regulate nitrate uptake and assimilation. The effects of treating barley roots with glutamine have been examined, first to identify the transport system for the uptake of the amino acid and then to measure root NR activity and cellular pools of nitrate. Treating N replete roots with glutamine elicited a change in the cell membrane potential and the size of this response was concentration dependent. In addition, the size of the electrical change depended on the previous exposures of the root to glutamine and was lost after a few cycles of treatment. Whole root tissue pools of glutamine and phenylalanine increased when roots were incubated in a nutrient solution containing 10 mM nitrate and 1 mM glutamine. Treating roots with 1 mM glutamine increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 h of treatment. Parallel measurements of root nitrate reductase activity during treatment with 1 mM glutamine showed a decrease. These measurements provide evidence for feedback regulation on NR activity that result in changes in cytosolic nitrate activity. After 6 h in glutamine both root NR activity and cytosolic nitrate activity returned to pretreatment values, while tissue concentrations of glutamine and phenylalanine remained elevated. The data are discussed in terms of the mechanisms that are most likely to be responsible for the changes in cytosolic nitrate.

Amino Acid Transport Systems↗
PubMed1998

Salivary nitrate, nitrite and nitrate reductase activity in relation to risk of oral cancer in Egypt.

It has been suggested that nitrate and nitrite may play a role in the etiology of human oral cancer. We investigated whether salivary nitrate and nitrite and the activity of nitrate reductase (NRase) may affect the risk of oral cancer in Egypt, an area with high levels of environmental nitrosating agents. Levels of salivary nitrite (8.3 +/- 1.0 micrograms/ml) and nitrate (44 +/- 3.7 micrograms/ml) and activity of NRase (74 +/- 10 nmol/ml/min) were significantly (P < 0.05) higher in oral cancer patients (n = 42) compared to control Egyptian healthy individuals (n = 40, nitrite = 5.3 +/- 0.3 micrograms/ml, nitrate = 27 +/- 1.2 micrograms/ml, and NRase activity = 46 +/- 4 nmol/ml/min). The adjusted odds ratio (OR) and the 95% confidence intervals (C.I.) for risk of oral cancer, categorized by the levels of salivary nitrate and nitrite and NRase activity, showed a higher cancer risk associated with nitrite > 7.5 micrograms/ml (OR: 3.0, C.I.: 1.0-9.3), nitrite > 40 micrograms/ml (OR: 4.3, C.I.: 1.4-13.3) and NRase activity > 50 nmol/ml/min (OR: 2.9, C.I.: 1.1-7.4). Our findings suggest that increased consumption of dietary nitrate and nitrite is associated with elevated levels of salivary nitrite. Together with the increased activity of salivary NRase, these observations may explain, at least in part, the role of nitrate and nitrite in the development of oral cancer in individuals from an area with a high burden of N-nitroso precursors.

Adult↗
PubMed2006

Impaired nitrate-mediated dilatation could reflect nitrate tolerance in patients with coronary artery disease.

AIM: To investigate whether nitrate-mediated dilatation (NMD) of the brachial artery could be impaired by the use of nitrates and reflect nitrate tolerance in coronary artery disease (CAD) patients. METHODS: A total of 243 patients (mean age 61+/-12 years, 146 male) were recruited before coronary angiography. Gensini score was calculated to assess the severity of CAD. Flow-mediated dilatation (FMD) and NMD of the brachial artery were measured. RESULTS: NMD progressively decreased from subjects without CAD (control group, n=71) to non-nitrate users (n=64) and nitrate users (n=108) with CAD (16.2+/-6.01% versus 13.4+/-5.57% versus 9.57+/-4.52%, P<0.001 for all). Multiple linear regression analysis showed that age, FMD, diameter of the brachial artery, hypertension, and nitrate use were independent determinants of NMD. In subgroup analysis of patients with high Gensini score (>10), nitrate users (n=56, 9.00+/-4.00%) had a significant lower NMD than non-users (n=28, 12.4+/-5.45%, P<0.001), but no difference in other parameters including age, prevalence of hypertension, FMD, Gensini score and use of other medications. CONCLUSIONS: The use of nitrates independently impaired NMD in CAD patients. An impaired NMD could be a comprehensive index of nitrate tolerance in patients with CAD.

Adult↗