Information on EC 1.7.1.1 - nitrate reductase (NADH)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.7.1.1
-
RECOMMENDED NAME
GeneOntology No.
nitrate reductase (NADH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrite + NAD+ + H2O = nitrate + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrate reduction II (assimilatory)
-
-
Nitrogen metabolism
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
nitrite:NAD+ oxidoreductase
An iron-sulfur molybdenum flavoprotein.
CAS REGISTRY NUMBER
COMMENTARY hide
9013-03-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Ankistrodesmus braunii
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain 750 and IM-43B
-
-
Manually annotated by BRENDA team
strain 750 and IM-43B
-
-
Manually annotated by BRENDA team
variant RH-30
-
-
Manually annotated by BRENDA team
italica
-
-
Manually annotated by BRENDA team
tea
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Berlin strain
-
-
Manually annotated by BRENDA team
Cucurbita sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
fragment; strain JCM11502, gene niaD
UniProt
Manually annotated by BRENDA team
fragment; strain JCM11502, gene niaD
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
cultivar Jessy
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strains H37Rv and RVW3
-
-
Manually annotated by BRENDA team
enzyme activity under different conditions, stability and activity of enzyme is not affected by reduced nitrogen sources
-
-
Manually annotated by BRENDA team
Pisum arvense
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
E1F1
-
-
Manually annotated by BRENDA team
expression in Pichia pastoris
Swissprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
var. macrocarpa
-
-
Manually annotated by BRENDA team
gene nasA
-
-
Manually annotated by BRENDA team
gene nasA
-
-
Manually annotated by BRENDA team
cultivar Ofanto
-
-
Manually annotated by BRENDA team
Ulva sp.
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 ferricyanide + NADH
2 ferrocyanide + NAD+ + H+
show the reaction diagram
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
show the reaction diagram
2,6-dichlorophenolindophenol + NADH
? + NAD+
show the reaction diagram
benzoquinone + NADH
benzoquinol + NAD+
show the reaction diagram
-
-
-
-
?
bromophenol blue + nitrate
? + nitrite
show the reaction diagram
Cucurbita sp.
-
-
-
-
?
FADH2 + nitrate
FAD + nitrite
show the reaction diagram
flavin hydroquinone + nitrate
? + nitrite
show the reaction diagram
-
-
-
-
?
FMNH2 + nitrate
FMN + nitrite
show the reaction diagram
menadione + NADH
reduced menadione + NAD+
show the reaction diagram
-
-
-
-
?
methylene blue + NADH
reduced methylene blue + NAD+
show the reaction diagram
-
-
-
-
?
NADH + BrO3-
NAD+ + ?
show the reaction diagram
-
-
-
-
?
NADH + ClO3-
NAD+ + ?
show the reaction diagram
-
-
-
-
?
NADH + IO3-
NAD+ + ?
show the reaction diagram
-
-
-
-
?
NADH + nitrate + H+
NAD+ + H2O + nitrite
show the reaction diagram
-
-
-
-
?
NADH + nitrate + H+
NAD+ + nitrite + H2O
show the reaction diagram
-
-
-
-
?
nitrate + NADH
nitrite + NAD+ + H2O
show the reaction diagram
nitrate + NADH + H+
nitrite + NAD+ + H2O
show the reaction diagram
nitrate + NADPH
nitrite + NADP+ + H2O
show the reaction diagram
nitrite + NADH
nitric oxide + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
reduced bromophenol blue + nitrate
bromophenol blue + nitrite
show the reaction diagram
reduced flavin + nitrate
? + nitrite
show the reaction diagram
-
-
-
-
?
reduced methyl viologen + nitrate
methyl viologen + nitrite
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
nitrate + NADH
nitrite + NAD+ + H2O
show the reaction diagram
-
the enzyme catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants
-
?
nitrate + NADH + H+
nitrite + NAD+ + H2O
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
activates
EDTA
-
5 mM, 27% increase of activity at pH 7.5
Mo6+
-
contains one Mo6+ per subunit
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%
Sodium azide
-
1 mM, 50% stimulation of menadione reduction
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
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
-
14-3-3B protein
-
-
-
2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid
3-(3,4-dichlorophenyl)-1,1-dimethylurea
-
inhibits the post-translational light activation of nitrate reducate
adenine
-
inhibition of the recombinant FAD domain
adenosine
-
inhibition of the recombinant FAD domain
adenosine 5'-diphosphoribose
Aminooxyacetate
-
-
AMP
-
inhibition of the recombinant FAD domain
ATP
-
at low pH, the presence of ATP alone in the incubation medium is sufficient to inactivate nitrate reductase
azide
Bromophenol blue
Cucurbita sp.
-
noncompetitive versus NADH
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
Carbamoyl phosphate
-
competitive
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
Co2+
-
1 mM, strong inhibition
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
Cyanate
-
-
cyanide
dicoumarol
-
competitive towards NADH
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
Fe3+
-
less potent inhibitor, effect cannot reversed by EDTA
ferricyanide
-
inhibition of the recombinant FAD domain
ferrocytochrome c
-
inactivation in a biphasic reaction, immune to inactivation during turnover with nitrate
glutamine
-
1 mM, decrease of activity
hydroxylamine
KCN
-
0.1 mM, complete inhibition
menadione
-
inhibits nitrate reductase
methyl methanethiosulfonate
MgCl2
MoO42-
-
1 mM, strong inhibition
mutant G216S of 14-3-3A protein
-
-
-
N-ethylmaleimide
-
1 mM, complete inhibition
NADPH
-
nitrate protects
NaN3
-
0.1 mM, complete inhibition
Ni2+
-
up to 0.1 mM, not inhibitory, 1 mM, 20% residual activity
nicotinamide
-
inhibition of the recombinant FAD domain
nitrite
NMN
-
inhibition of the recombinant FAD domain
NO2-
-
10 mM, 65% inhibition
O-Methoxylamine
-
-
o-phenanthroline
-
1 mM, complete inhibition
p-chloromercuribenzoic acid
-
0.01 mM, complete inhibition
p-hydroxymercuribenzoate
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
pyridoxal 5'-phosphate
Thiocyanate
VO3-
-
1 mM, strong inhibition
Zn
-
after 24 h activity is reduced by 11%, 19% and 21% at 1 mM, 10 mM and 100 mM of Cr, respectively
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethylamine
-
5 mM, 5.6fold stimulation
Calmodulin
-
activates
EDTA
-
reduction of nitrate and menadione requires EDTA
GSH
-
5 mM, 3.8fold stimulation
HPO42-
-
activates
L-Cys
-
5 mM, 5.6fold stimulation
L-cysteic acid
-
5 mM, 2fold stimulation
L-cysteine methyl ester
-
5 mM, 5.6fold stimulation
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
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
100
2,6-dichlorophenol indophenol
-
-
0.053 - 0.13
2,6-dichlorophenolindophenol
0.739
BrO3-
-
ionic strength: 50 mM
0.06
Bromophenol blue
Cucurbita sp.
-
at 10 mM nitrate
0.221
ClO3-
-
ionic strength: 50 mM
0.0026
FAD
-
-
0.021 - 0.045
ferricyanide
0.006 - 80
ferricytochrome c
0.056
flavin hydroquinone
-
ionic strength: 50 mM or 200 mM
-
10.75
IO3-
-
ionic strength: 50 mM
0.275
menadione
-
-
0.0029 - 0.067
NADH
0.026
NADPH
-
-
0.008 - 13
nitrate
0.54
reduced bromophenol blue
-
-
-
0.87
reduced methyl viologen
-
-
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
835 - 1300
ferricyanide
1.5
ferricytochrome c
Spinacia oleracea
-
pH 7.0, 25C, functional FAD-containing domain plus heme-containig domain
1.4 - 1400
NADH
1.8 - 33
nitrate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00034
14-3-3 protein isoform chi
-
22C, pH not specified in the publication
-
0.00014
14-3-3 protein isoform kappa
-
22C, pH not specified in the publication
-
0.00008
14-3-3 protein isoform lambda
-
22C, pH not specified in the publication
-
0.00023
14-3-3 protein isoform ni
-
22C, pH not specified in the publication
-
0.00006
14-3-3 protein isoform omega
-
22C, pH not specified in the publication
-
0.00031
14-3-3 protein isoform phi
-
22C, pH not specified in the publication
-
0.00076
14-3-3 protein isoform theta
-
22C, pH not specified in the publication
-
0.0000015 - 0.0000017
14-3-3B protein
-
26
adenine
-
inhibition of the recombinant FAD domain
4.8
adenosine
-
inhibition of the recombinant FAD domain
0.2 - 1.36
ADP
0.6
AMP
-
inhibition of the recombinant FAD domain
0.3
Bromophenol blue
Cucurbita sp.
-
-
0.018
Carbamoyl phosphate
-
-
15 - 176
Cl-
0.013
dicoumarol
-
nitrate reductase assay
23
ferricyanide
-
inhibition of the recombinant FAD domain
0.12
hydroxylamine
Ankistrodesmus braunii
-
-
0.0000049 - 0.0000054
mutant G216S of 14-3-3A protein
-
0.5 - 2
NAD+
122
nicotinamide
-
inhibition of the recombinant FAD domain
62
NMN
-
inhibition of the recombinant FAD domain
0.176
pyridoxal 5'-phosphate
-
-
additional information
additional information
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
-
in leaves of nitrate-free grown plants
0.0015
-
His-tagged molybdenum domain, 25C, pH 7.6
0.0108
-
GST-tagged molybdenum domain, 25C, pH 7.6
0.029
-
in leaves of plants grown on medium containing 10 mM nitrate
0.03
-
in roots of nitrate-free grown plants
0.045
-
in roots of plants grown on medium containing 10 mM nitrate
0.89
-
-
9.51
-
c2NR
10.5
cells in presence of NO3- and under O2-limitation; cells in presence of NO3- and under O2-limitation
23.5
-
-
52.33
-
pH 8.5, 25C
60 - 70
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
assay at; assay at
7.3
-
NADH:cytochrome c reductase activity
7.3 - 7.5
-
-
7.3
-
NADH:cytochrome c reductase activity
7.9
-
NADH:nitrate reductase activity
9
-
NADH-nitrate reductase activity
9.5
-
methyl viologen-nitrate reductase activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6.5
-
in MES buffer
6 - 8.5
-
pH 6.0: about 35% of maximal activity, pH 8.5: about 45% of maximal activity
6.5 - 8
-
in 0.05 mM MOPS buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15
-
enzyme from cells grown at 5C, 15C or at 25C
25
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-10 - 40
-
-10C: 8% of maximal activity, 0C: 23% of maximal activity, 40C: 19% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
-
Manually annotated by BRENDA team
-
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
Manually annotated by BRENDA team
additional information
-
guard cell, generating nitric oxide in response to treatment with abscisic acid and nitrite
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
91000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
144000
-
non-denaturing pore gradient PAGE
147000
-
gel filtration
180000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
202000
205000
-
non-denaturing PAGE
220000
-
gel filtration
221000
-
sucrose density gradient centrifugation, gel filtration
225000
-
disc gel electrophoresis
230000
-
disc gel electrophoresis
270000
-
gel filtration, sucrose density gradient centrifugation
280000
-
equilibrium sedimentation
330000
-
gel filtration
356000
-
sucrose density gradient centrifugation, gel filtration
360000
-
equilibrium sedimentation
362000
-
4 active fractions with MW of 91000 Da, 362000 Da, 180000 Da and 720000 Da are detected, gel filtration
440000
-
gel filtration
514000
-
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
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
-
6 * 85000, SDS-PAGE
homodimer
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
additional information
-
contains little or no carbohydrate; contains no or little lipid
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
-
1 h, stable
392884
additional information
-
activity determined at pH 6.0 was significantly lower than at pH 7.5
672500
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
pH 7.5, half-life: 42 h
10
-
half-life: 94 min
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 0C and for several months at -80C
-
FAD protects from heat inactivation at 45C
-
FAD stabilizes at 25C, at 10C FAD has no effect on stability
-
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
-
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 0C and at 25C
presence of casein in phosphate buffer improves stability at 0C but not at 30C
-
stabilized at 0C and at 30C 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
-
stabilized in vitro by addition of chymostatin to extraction buffer
-
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
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Glycerol
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50 mM MOPS, containing 0.01 mM FAD, 0.1 mM EDTA, pH 7.0, 50% glycerol, stable for several months
-
-20C, pH 6.9, phosphate biuffer, 40% v/v glycerol, stable for more than 2 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation and Ble Sepharose chromatography
-
by affinity chromatography using 5'AMP Sepharose and monoclonal antibodies
-
extracting strength of different detergents on membrane-bound isozyme
-
flavin domain
HiTrap Q column anion-exchange chromatography
-
immunoaffinity chromatography using a monoclonal antibody
-
mutant enzymes K741R, K741H, K714A, K741E, K741M, K741Q, K741P
-
NADH:Fe(III)-citrate reductase activity copurifies and has identical electrophoretic mobility
-
partial
recombinant enzyme expressed in Pichia pastoris
-
removal of nicked subunits by affinity chromatography
-
Sephadex G-25 gel filtration
-
SourceQ15 column chromatography and Ni-NTA column chromatography
-
the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in Escherichia coli
-
wild-type and nr1-mutant
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Nicotiana plumbaginifolia
-
expression in Pichia pastoris
-
expression of amino-terminal, molybdenum-containing domain in Escherichia coli either as His-tagged or GST-tagged fusion protein
-
gene nar1, genotyping of wild-type and mutant nar1 enzyme, overview
-
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
mutant S534A is expressed in Pichia pastoris strainKM71
-
the C-terminal 268 residues corresponding to the flavin-containing domain, amplified and expressed in Escherichia coli
-
two homologous genes coding for nitrate reductase in tobacco
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C191A
-
enzyme is still produced, but it is inactive
C191S
-
enzyme is still produced, but it is inactive
S266A
-
48% of the activity of wild-type enzyme
S534A
-
the mutant is not inhibited by 14-3-3 proteins
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
C240A
-
decrease of diaphorase activity
C240G
-
decrease of diaphorase activity
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
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