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Enzyme Nomenclature
Information on EC 1.7.1.1 - nitrate reductase (NADH)
Word Map on EC 1.7.1.1
- 1.7.1.1
- chlorella
- molybdenum
-
nadph-nitrate
-
nadh:cytochrome
-
1.6.6.2
-
nitrate-reducing
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
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EC NUMBER 
COMMENTARY 
1.7.1.1
-
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RECOMMENDED NAME
GeneOntology No.
nitrate reductase (NADH)
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nitrite + NAD+ + H2O = nitrate + NADH + H+
catalytic rate is determined by a combination of rates with no overall rate limiting individual process
-
nitrite + NAD+ + H2O = nitrate + NADH + H+
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
nitrite:NAD+ oxidoreductase
An iron-sulfur molybdenum flavoprotein.
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CAS REGISTRY NUMBER
COMMENTARY
9013-03-0
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
enzyme activity under different conditions, stability and activity of enzyme is not affected by reduced nitrogen sources
-
-
rapid inactivition/activation of enzyme in response to dark/light transition
-
-
cv. Mill, membrane-bound isozyme remains more active in the dark and is more easily recovered after transfer to light than cytosolic isoform, inhibition of both forms by saline stress, cytosolic form is strongly induced and strictly dependent on presence of nitrate
-
-
-
392837, 392843, 392852, 392853, 392859, 392862, 392870, 392871, 392872, 392880, 392882, 392884, 392886, 392891, 392897, 392899, 392902, 392903, 392916, 392917, 654345
-
-
expression of amino-terminal, molybdenum-containing domain in Escherichia coli either as His-tagged or GST-tagged fusion protein
-
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
-
-
-
-
?
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
-
-
-
-
?
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
392837, 392843, 392852, 392853, 392859, 392862, 392870, 392871, 392872, 392880, 392882, 392884, 392886, 392891, 392897, 392899, 392902, 392903, 392915, 392916, 392917
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
the enzyme catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
-
-
287994, 392845, 392855, 392865, 392869, 392876, 392878, 392883, 392886, 392891, 392893, 392894, 392900
-
-
?
nitrate + NADH + H+
nitrite + NAD+ + H2O
the enzyme is involved in the reduction of NO3- to nitrite at the initial step of denitrification
-
-
?
nitrate + NADH + H+
nitrite + NAD+ + H2O
the enzyme is involved in the reduction of NO3- to nitrite at the initial step of denitrification
-
-
?
reduced bromophenol blue + nitrate
bromophenol blue + nitrite
-
-
-
-
?
reduced bromophenol blue + nitrate
bromophenol blue + nitrite
-
-
-
-
?
reduced methyl viologen + nitrate
methyl viologen + nitrite
-
-
-
-
?
reduced methyl viologen + nitrate
methyl viologen + nitrite
-
-
-
-
-
reduced methyl viologen + nitrate
methyl viologen + nitrite
-
-
-
-
?
reduced methyl viologen + nitrate
methyl viologen + nitrite
-
-
-
-
?
additional information
?
-
-
partial activities reside on functionally independent domains
-
-
-
additional information
?
-
-
the enzyme could have a role in iron assimilation
-
-
-
additional information
?
-
-
no activity with coenzyme Q10 and plastoquinone
-
-
-
additional information
?
-
-
in extracts of algae grown under either constant dimlight or light-dark cycle, the activity of nitrate reductase exhibits a daily rhythm, peaking at midday phase as does photosynthesis
-
-
-
additional information
?
-
-
key enzyme of nitrogen metabolism in higher plants
-
-
-
additional information
?
-
-
minor role of enzyme in the regulation of nitrate assimilation pathway
-
-
-
additional information
?
-
-
effect of nitrate, ammonium, light and a plastidic factor on the appearance of multiple forms of nitrate reductase
-
-
-
additional information
?
-
-
the isolated flavin-containing domain is capable of reducing cytochrome b5 directly
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
nitrate + NADH
nitrite + NAD+ + H2O
-
the enzyme catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants
-
?
nitrate + NADH + H+
nitrite + NAD+ + H2O
B1B5E3, B1B5E4
the enzyme is involved in the reduction of NO3- to nitrite at the initial step of denitrification
-
-
?
nitrate + NADH + H+
nitrite + NAD+ + H2O
B1B5E3, B1B5E4
the enzyme is involved in the reduction of NO3- to nitrite at the initial step of denitrification
-
-
?
additional information
?
-
-
the enzyme could have a role in iron assimilation
-
-
-
additional information
?
-
-
in extracts of algae grown under either constant dimlight or light-dark cycle, the activity of nitrate reductase exhibits a daily rhythm, peaking at midday phase as does photosynthesis
-
-
-
additional information
?
-
-
key enzyme of nitrogen metabolism in higher plants
-
-
-
additional information
?
-
-
minor role of enzyme in the regulation of nitrate assimilation pathway
-
-
-
additional information
?
-
-
effect of nitrate, ammonium, light and a plastidic factor on the appearance of multiple forms of nitrate reductase
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
-
molybdenum, heme and FAD components are localized in distinct domains which are covalently linked by exposed hinge regions
heme
-
molybdenum, heme and FAD components are localized in distinct domains which are covalently linked by exposed hinge regions
NADH
-
-
392837, 392843, 392852, 392853, 392859, 392862, 392870, 392871, 392872, 392880, 392882, 392884, 392886, 392891, 392897, 392899, 392902, 392903, 392915, 392916, 392917, 654345
NADH
-
-
287994, 392845, 392855, 392865, 392869, 392876, 392878, 392883, 392886, 392891, 392893, 392894, 392900, 392907, 674455
NADH
-
-
additional information
-
the enzyme is associated with a cytochrome of the b type
-
additional information
-
no measurable activity in presence of NADPH
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe
Molybdenum
NaCl
-
100 mM NaCl increases enzyme activity in leaves from plants grown on 0.1 or 1 mM nitrate about 3fold
Ni2+
-
a significant improvement of activity is achieved after cyanide treatment by addition of 5 mM NiCl2 which increases the enzyme recovery in leaf extract up to 63%
additional information
Molybdenum
-
gamma subunit which carries molybdenum-containing component of 1000 Da
Molybdenum
-
molybdenum, heme and FAD components are localized in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex
Molybdenum
-
oxidation-reduction midpoint potentials of the molybdenum center: -8 mV for the Mo(VI)/Mo(V) couple and -42 mV for the Mo(V)/Mo(IV) couple
Molybdenum
-
Mo/protein ratio 0.44 for His-tagged protein, 0.93 for GST-tagged protein
additional information
-
increased ionic strength stimulates NADH:nitrate reductase activity
additional information
-
tungsten cannot replace for molybdenum in the GST-tagged protein
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
14-3-3 protein BMH1
-
0.0018 mM, reduces activity to less than 20%, at pH 6.0
-
14-3-3 protein isoform chi
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform epsilon
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform kappa
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform lambda
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform ni
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform omega
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform phi
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
14-3-3 protein isoform theta
-
noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme
-
3-(3,4-dichlorophenyl)-1,1-dimethylurea
-
inhibits the post-translational light activation of nitrate reducate
ATP
-
at low pH, the presence of ATP alone in the incubation medium is sufficient to inactivate nitrate reductase
Ca2+
-
5 mM, 90% inhibition of the low activity form, no activity of high activity form, inhibition is prevented by low concentrations of thiol compounds
Cd2+
-
0.002 mM, 40% inhibition, 10 mM EDTA protects up to 0.1 mM metal concentration
Cl-
-
alters the observed Mo(V) lineshape, mixed-type inhibitor, decreases both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities
Cr
-
after 24 h activity is reduced by 17.31%, 30.72% and 45% at 1 mM, 10 mM and 100 mM of Cr, respectively
Cr3+
-
0.002 mM, 20% inhibition, 10 mM nitrilotriacetic acid does not protect
Cr6+
-
0.001 mM, 62% inhibition, 10 mM nitrilotriacetic acid does not protect
Cu
-
after 24 h activity is reduced by 21.5%, 36% and 46% at 1 mM, 10 mM and 100 mM of Cu, respectively
dithiothreitol
-
rate of inactivation is increased by NAD+, but not by NADP+
Fe2+
-
potent inhibitor, inhibition can be abolished by prior chelation of the metal by EDTA
ferrocytochrome c
-
inactivation in a biphasic reaction, immune to inactivation during turnover with nitrate
phosphate
-
25 mM, increases activity with a nitrate concentration of 2 mM, decreases activity with a nitrate concentration of 0.1 mM
potassium ferricyanide
-
preincubation with potassium ferricyanide inactivates nitrate reductase
Zn
-
after 24 h activity is reduced by 11%, 19% and 21% at 1 mM, 10 mM and 100 mM of Cr, respectively
Cu2+
-
0.001 mM, 76% inhibition, 10 mM EDTA protects up to 0.1 mM metal concentration
Cu2+
-
potent inhibitor, inhibition can be abolished by prior chelation of the metal by EDTA
cyanide
-
mechanism of reactivation of cyanide-inactivated nitrate reductase by flavins in light
cyanide
-
reactivation by incubation with oxidant systems after inactivation by treatment with NADH and cyanide
cyanide
-
inactivation by simultaneous presence of NADH and low concentrations of cyanide, reactivation by incubation with ferricyanide or by a short exposure to light in the presence of FAD
hydroxylamine
Ankistrodesmus braunii
-
NO3-, cyanate, carbamoyl phosphate and azide protect from inactivation. Photoreactivation in presence of flavins, early inhibition appears to be competitive versus NO3-
hydroxylamine
-
interacts with reduced cytochrome b557 during catalysis of the enzyme
Mg2+
-
5 mM MgCl2 decreases enzyme activity at pH 7.5, this effect is completely reversed by the addition of EDTA into samples returning the enzyme's activity to its initial level
Mg2+
-
5 mM, 70% inhibition of the low activity form, no inhibition of high activity form, inhibition is prevented by low concentrations of thiol compounds
Mg2+
native protein, highly sensitive to Mg2+, recombinant protein, not sensitive. Recombinant protein plus enzyme-free leaf extract of Ricinus communis shows restored high sensitivity to Mg2+, but remains unresponsive to ATP
NaCl
-
100 mM NaCl reduces enzyme activity in leaves from plants grown on 10 mM nitrate and in roots from plants grown on 0.1 mM nitrate to about 50%
NAD+
-
inactivation in presence of thiol compounds is enhanced by cyanide ions and can be reversed by ferricyanide
NADH
-
inactivation in a biphasic reaction, immune to inactivation during turnover with nitrate
NADH
-
p-hydroxymercuribenzoate causes the appearance of an FAD-requirement for inactivation by NADH of FMNH2-nitrate reductase
NADH
-
conversion of the enzyme to a reduced inactive form, by preincubation with NADH, in absence of nitrate, occurs in presence of either dithiothreitol and/or FAD but not with cysteine. Pretreatment with NADH alone does not inactivate, a nucleophilic agent, i.e. cyanide or superoxide is necessary to inhibit electron transfer by the enzyme to nitrate
NADH
-
0.01 mM NADH, in absence of nitrate, 50% loss of activity after 30 min, 0.05 mM nitrate prevents inactivation, 0.001 mM cyanide enhances degree of inactivation. Rapid reactivation after treatment with 0.3 mM ferricyanide or exposure to light, 230 mWatt per cm2, plus 0.02 mM flavin adenine dinucleotide
p-hydroxymercuribenzoate
Cucurbita sp.
-
inactivation of NADH:nitrate reductase activity, no loss of bromphenol blue: nitrate reductase activity
p-hydroxymercuribenzoate
-
inhibition of full and NADH-utilizing partial activities
Pb2+
-
0.002 mM, 60% inhibition, 10 mM EDTA protects up to 0.1 mM metal concentration
Zn2+
-
0.004 mM, 60% inhibition, 10 mM EDTA protects up to 0.1 mM metal concentration
Zn2+
-
potent inhibitor, inhibition can be abolished by prior chelation of the metal by EDTA
additional information
-
increasing level of salinity causes decrease in enzyme activity
-
additional information
-
An inhibitory effect of Mg-ATP is cancelled in the presence of staurosporine (the protein kinase inhibitor) and completely reversed after addition of EDTA as well as AMP
-
additional information
-
inhibition by monospecific anti-nitrate reductase rabbit serum
-
additional information
-
purification of a NADH-nitrate reductase inhibitor from young leaves of Glycine max, that causes a reversible inhibition
-
additional information
-
the 14-3-3A protein and the 14-3-3C protein are functionally not capable to inhibit nitrate reductase
-
additional information
-
roots of seedlings from Oryza sativa contain a substance which inhibits the activity of nitrate reductase when NADH or FMNH2 is used as electron donor
-
additional information
-
inhibitor from primary and regenerated roots of nitrate-grown seedlings, main site of action is NADH:cytochrome c reductase component of the nitrate reductase, NADH protects
-
additional information
-
nitrate reductase inhibitor from root extract of rice seedlings. Inactivation proceeds in two steps: the inhibitor first binds with nitrate reductase to cause a reduction in both NADH:nitrate reductase and reduced benzyl viologen:nitrate reductase activity. In the second phase, there is a complete inactivation of NADH:nitrate reductase after about 20 min. Reduced benzyl viologen:nitrate reductase activity is not affected by the second phase of inactivation
-
additional information
Pisum arvense
-
maize root inactivating enzyme inactivates pea leaf nitrate reductase
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
light
-
enzyme is posttranslationally activated by light. Light also has a positive effect also on de novo synthesis of nitrate reductase. Daily variations in nitrate reductase activity are mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis and stomata opening
-
phosphate
-
25 mM, increases activity with a nitrate concentration of 2 mM, decreases activity with a nitrate concentration of 0.1 mM
additional information
-
Co-cultivation of Arabidopsis seedlings with Piriformospora indica causes a 29.8% increase in the plant-specific NADH-dependent NR activity in the roots
-
additional information
-
Co-cultivation of tobacco seedlings with Piriformospora indica causes a 50.2% increase in the plant-specific NADH-dependent activity in the roots.
-
additional information
-
no activity recovering effect of Ni2+ application after cyanide treatment
-
additional information
-
enzyme does not reveal any effects of light/darkness on nitrate reductase activity state
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.027
ferricyanide
-
wild-type recombinant flavin domain of nitrate reductase
0.036
ferricyanide
-
recombinant flavin domain of nitrate reductase C54S mutant
0.045
ferricyanide
-
recombinant flavin domain of nitrate reductase C17S mutant
0.015
NADH
-
recombinant flavin domain of nitrate reductase C17S mutant; recombinant flavin domain of nitrate reductase C54S mutant
additional information
additional information
-
Km-values of the the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in E. coli
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.5
ferricytochrome c
Spinacia oleracea
-
pH 7.0, 25°C, functional FAD-containing domain plus heme-containig domain
1400
NADH
Spinacia oleracea
-
pH 7.0, 25°C, functional FAD-containing domain domain plus heme-containig domain
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00034
14-3-3 protein isoform chi
-
22°C, pH not specified in the publication
-
0.00014
14-3-3 protein isoform kappa
-
22°C, pH not specified in the publication
-
0.00008
14-3-3 protein isoform lambda
-
22°C, pH not specified in the publication
-
0.00023
14-3-3 protein isoform ni
-
22°C, pH not specified in the publication
-
0.00006
14-3-3 protein isoform omega
-
22°C, pH not specified in the publication
-
0.00031
14-3-3 protein isoform phi
-
22°C, pH not specified in the publication
-
0.00076
14-3-3 protein isoform theta
-
22°C, pH not specified in the publication
-
1.9
NAD+
-
recombinant flavin domain of nitrate reductase wild-type and C17S mutant
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.029
-
in leaves of plants grown on medium containing 10 mM nitrate
0.045
-
in roots of plants grown on medium containing 10 mM nitrate
10.5
cells in presence of NO3- and under O2-limitation; cells in presence of NO3- and under O2-limitation
additional information
additional information
enzyme activity in cells under different culture conditions, overview; enzyme activity in cells under different culture conditions, overview
additional information
enzyme activity in cells under different culture conditions, overview; enzyme activity in cells under different culture conditions, overview
additional information
-
enzyme activity in cells under different culture conditions, overview; enzyme activity in cells under different culture conditions, overview
additional information
-
in vivo nitrate reductase assay by vacuum-infiltration procedure in leaf sections
additional information
-
in vivo nitrate reductase assay by vacuum-infiltration procedure in leaf sections
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.5
7.5
8.5
6.5
-
NADH:ferricyanide reductase activity, NADH:dichlorophenolindophenol reductase activity and reduced methyl viologen:nitrate reductase activity
8.5
-
NADH-ferricyanide reductase activity and NADH-cytochrome c reductase activity
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 8.5
-
pH 6.0: about 35% of maximal activity, pH 8.5: about 45% of maximal activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
-10 - 40
-
-10°C: 8% of maximal activity, 0°C: 23% of maximal activity, 40°C: 19% of maximal activity
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
suspension culture of the XD cell line. As tobacco XD cell culture progresses through the exponential phase of growth into the stationary phase, due to depletion of nitrate, the overt nitrate reductase activity decreases and the latent form becomes predominant. 9fold activation of the enzyme activity is observed in extracts from late-exponential phase cultures
additional information
-
guard cell, generating nitric oxide in response to treatment with abscisic acid and nitrite
-
enzyme from leaf and root are different proteins on the basis of their antigenic behaviour
-
enzyme from leaf and root are different proteins on the basis of their antigenic behaviour
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
constitutive nitrate reductase exists mostly in membranes
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PDB
SCOP
CATH
ORGANISM
UNIPROT
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20000
-
alpha2beta2gamma2molybdenum-containing-component, 2 * 40000 Da FAD-containing alpha-subunit + 2 * 20000 Da cytochrome b557 beta subunit + 2 *gamma subunit which carries molybdenum-containing component of 1000 Da
40000
-
alpha2beta2gamma2molybdenum-containing-component, 2 * 40000 Da FAD-containing alpha-subunit + 2 * 20000 Da cytochrome b557 beta subunit + 2 *gamma subunit which carries molybdenum-containing component of 1000 Da
55000
-
x * 55000, SDS-PAGE, His-tagged domain, x * 85000, SDS-PAGE, GST-tagged domain
85000
90000
91000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
100000
110000
180000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
202000
362000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
720000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
additional information
-
the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in E. coli show a MW of 30000 Da by SDS-PAGE
85000
-
x * 55000, SDS-PAGE, His-tagged domain, x * 85000, SDS-PAGE, GST-tagged domain
90000
-
x * 90000, equilibrium sedimentation of enzyme dissociated in 6 M guanidine hydrochloride
100000
-
4 * 100000, at low protein concentration, the tetramer dissociates to a fully active dimer, each subunit in the tetramer or dimer can function independently, radiation inactivation analysis
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
tetramer
additional information
?
-
x * 90000, equilibrium sedimentation of enzyme dissociated in 6 M guanidine hydrochloride; x * 98000, SDS-PAGE
?
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alpha2beta2gamma2molybdenum-containing-component, 2 * 40000 Da FAD-containing alpha-subunit + 2 * 20000 Da cytochrome b557 beta subunit + 2 *gamma subunit which carries molybdenum-containing component of 1000 Da
?
-
x * 55000, SDS-PAGE, His-tagged domain, x * 85000, SDS-PAGE, GST-tagged domain
tetramer
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4 * 100000, at low protein concentration, the tetramer dissociates to a fully active dimer, each subunit in the tetramer or dimer can function independently, radiation inactivation analysis
additional information
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FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, the quarternary structure maintains via association sites on the heme/molybdenum domain
additional information
-
two bands of 107000 Da and 99500 Da are detected by SDS-PAGE
additional information
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diaphorase activity is located in the small subunit
additional information
-
diaphorase activity is located in the small subunit
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
phosphoprotein
additional information
-
contains little or no carbohydrate; contains no or little lipid
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
additional information
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activity determined at pH 6.0 was significantly lower than at pH 7.5
672500
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
30
40
30
-
30-50% (NH4)2SO4, 150 mM potassium phosphate buffer, pH 7.5, 0.01 mM FAD, 1 mM dithiothreitol, half-life: 36.8 min
40
Cucurbita sp.
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40 min, 50% loss of nitrate reductase activity, slight increase of bromphenol blue activity
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
0.03% SDS produces almost complete inactivation of NADH-diaphorase and NADH-nitrate reductase, while FNH2-nitrate reductase retains 60% of the original activity, FAD has no protecting effect
-
1 M guanidine hydrochloride, in presence of FAD, 80% inactivation of FNH2-nitrate reductase activity and NADH-nitrate reductase activity, NADH-diaphorase activity is unaffected
-
4 M urea, in presence of FAD, inactivation of FMNH2-nitrate reductase and NADH-nitrate reductase activity, only a slight effect on the NADH-diaphorase activity
-
dilution of a crude extract leads to increasing lability, much more stable in presence of both NADH and nitrate
-
enzyme in the crude extract is stable for several days at 0°C and for several months at -80°C
-
imposition of water stress or artificial extension of the dark period leads to significant reduction of nitrate reductase activity, but does not change in vitro nitrate reductase stability
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inactivation by corn root proteinase, comparison of hydrolysis products
incubation of the native enzyme with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in loss of NADH:nitrate reductase and NADH:cytochrome c reductase activity, but no loss of methyl viologen:nitrate reductase activity
-
leupeptin inhibits degradation of NADH-nitrate reductase by thiol-dependent acid endoproteinase in primary leaf extract
-
preincubation with NADH stabilizes activity at 0°C and at 25°C
presence of casein in phosphate buffer improves stability at 0°C but not at 30°C
-
stabilized at 0°C and at 30°C by buffer containing 0.25 M Tris-HCl, pH 8.5, 3 mM DTT, 0.005 mM FAD, 0.001 mM sodium molybdate and 1 mM EDTA
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the decay rate of nitrate reductase activity in crude extracts is due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0-30% and the other in the 50-70% saturated (NH4)2SO4 fraction of the crude extract
-
the enzyme is very susceptible to inactivation by maize root proteinase and trypsin
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Glycerol
Glycerol
enzyme activity is strongly negatively impacted by increase in solution viscosity using 50% glycerol
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50 mM MOPS, containing 0.01 mM FAD, 0.1 mM EDTA, pH 7.0, 50% glycerol, stable for several months
-
-20°C, pH 6.9, phosphate biuffer, 40% v/v glycerol, stable for more than 2 months
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by affinity chromatography using 5'AMP Sepharose and monoclonal antibodies
-
NADH:Fe(III)-citrate reductase activity copurifies and has identical electrophoretic mobility
-
partial
the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of amino-terminal, molybdenum-containing domain in Escherichia coli either as His-tagged or GST-tagged fusion protein
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gene nasA, DNA and amino acid sequence determination and analysis, sequence comparisons and detailed phylogenetic analysis, overview
gene niaD, DNA and amino acid sequence determination and analysis, functional complemention of the defective NO3- assimilation by mutant strain M10, expression in strain T1; gene niaD, DNA and amino acid sequence determination and analysis, functional complemention of the defective NO3- assimilation by mutant strain M10, expression in strain T1
high-level expression of the catalytically active flavin domain in Escherichia coli
the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in Escherichia coli
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gene nasA, DNA and amino acid sequence determination and analysis, sequence comparisons and detailed phylogenetic analysis, overview
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gene nasA, DNA and amino acid sequence determination and analysis, sequence comparisons and detailed phylogenetic analysis, overview
-
gene nasA, DNA and amino acid sequence determination and analysis, sequence comparisons and detailed phylogenetic analysis, overview
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S521D
C17S
-
visible and CD spectra are very similar to that of the wild-type domain, thermal stability is slightly decreased compared to the wild-type domain
C240S
-
decrease of diaphorase activity, thermal stability is slightly decreased compared to the wild-type domain, the oxidation reduction midpoint potential for the FAD/FADH2 couple is -219 mV compared to -268 mV in the wild-type domain
C54S
-
visible and CD spectra are very similar to that of the wild-type domain, thermal stability is slightly decreased compared to the wild-type domain,the oxidation reduction midpoint potential for the FAD/FADH2 couple is -197 mV compared to -268 mV in the wild-type domain
C62S
-
visible and CD spectra are very similar to that of the wild-type domain, thermal stability is decreased compared to the wild-type domain, the oxidation reduction midpoint potential for the FAD/FADH2 couple is -226 mV compared to -268 mV in the wild-type domain
K714A
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741E
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741H
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741M
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741P
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741Q
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
K741R
-
functional flavoprotein which retains FAD as the sole prosthetic group and exhibits spectroscopic properties comparable to that of the wild-type enzyme. Altered NADH:ferricyanide reductase activity with mutations affecting both turnover number and the Km-value for NADH. At pH 7.0 the turnover number decreases in the order wild-type, K741R, K741A, K741H, K741E, K741M, K741Q. Km-value for NADH increases in the same order
additional information
S521D
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complete abolishment of inactivation in response to light/dark transition rendering enzyme in activated form. Upon high nitrate concentrations, transgenic plants accumulate nitrite in darkness and young leaves show chlorosis
S521D
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the protein is constitutively active under conditions where the wild type protein would be rapidly inactivated, such as in the dark
additional information
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ammonia levels in the wild-type are decreased by about one-half by CO2 enrichment, whereas ammonia is unaffected by elevated CO2 in nar1 mutant leaves
additional information
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expression of fragment A542-Y647 in Escherichia coli, production of enzyme heme domain evidenced by pink cells. Reconstitution of –NADH:cytochrome c reductase activty in presence of recombinant form of spinach nitrate reductase flavin domain
additional information
-
expression of 541 residue amonio-terminal, molybdenum-containing domain in Escherichia coli either as His-tagged or GST-tagged fusion protein
LINKS TO OTHER DATABASES (specific for EC-Number 1.7.1.1)
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