Information on EC 1.7.1.3 - nitrate reductase (NADPH)

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

EC NUMBER
COMMENTARY hide
1.7.1.3
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RECOMMENDED NAME
GeneOntology No.
nitrate reductase (NADPH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nitrite + NADP+ + H2O = nitrate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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redox reaction
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-
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
nitrate reduction V (assimilatory)
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Nitrogen metabolism
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
nitrite:NADP+ oxidoreductase
An iron-sulfur molybdenum flavoprotein.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-28-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
Lupinus, strain USDA 3045
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Manually annotated by BRENDA team
Hedw.
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
nar1; symbiotic basidomycete, 3 wild-type haploid strains, progenies of the HC1 dikaryotic strain GCA6, gene nar1
SwissProt
Manually annotated by BRENDA team
L. cv. Steptoe nar1a and nar1a; nar7w genotypes
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Manually annotated by BRENDA team
5297a
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Manually annotated by BRENDA team
STA4
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Manually annotated by BRENDA team
gene tpnr1
SwissProt
Manually annotated by BRENDA team
gene tbnr1, ascomycete
SwissProt
Manually annotated by BRENDA team
gene tbnr1, ascomycete
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization, molybdenum cofactor-dependent enzyme maturation, overview
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 + NADPH
2 ferrocyanide + NADP+ + H+
show the reaction diagram
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-
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?
chlorate + NADPH
chlorite + NADP+
show the reaction diagram
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lower affinity than to nitrate, appears to be toxic or its product
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?
NADPH + nitrate
NADP+ + nitrite
show the reaction diagram
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-
-
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?
nitrate + NADPH
nitrite + NADP+
show the reaction diagram
nitrate + NADPH
nitrite + NADP+ + H2O
show the reaction diagram
nitrate + NADPH + H+
nitrite + NADP+
show the reaction diagram
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-
-
?
nitrate + NADPH + H+
nitrite + NADP+ + H2O
show the reaction diagram
nitrate + reduced benzyl viologen
nitrite + benzyl viologen
show the reaction diagram
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-
-
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?
nitrate + reduced methyl viologen
nitrite + oxidized methyl viologen
show the reaction diagram
additional information
?
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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 + NADPH
nitrite + NADP+
show the reaction diagram
nitrate + NADPH
nitrite + NADP+ + H2O
show the reaction diagram
nitrate + NADPH + H+
nitrite + NADP+ + H2O
show the reaction diagram
additional information
?
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Q9UVH6
enzyme expression is regulated by different inorganic and organic nitrogen sources, e.g. nitrate, ammonium, urea and glutamate, regulatory mechanism of nitrate aquisition in ectomycchorizae, overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
benzyl viologen
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cytochrome b557
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cytochrome c
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FMN
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to a lesser extent than FAD
molybdenum cofactor
molybdopterin
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i.e Moco/MPT, binding of molybdenum cofactor to apo-nitrate reductase is independent from other prosthetic groups, molybdenum cofactor-dependent enzyme maturation, overview. Reconstitution of Moco-free nitrate reductase with various amounts of purified Moco carrier protein
NADPH
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mo5+
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in the active site-bound molybdenum cofactor
Molybdenum
NH4+
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represses the intracellular NO3- assimilation, which is required for the induction and maintenace of NADPH nitrate reductase, enzyme is regulated by NH4+ at the transcriptional level
phosphate
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stimulates, required for maximal activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8-hydroxyquinoline
azide
Cu2+
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inhibitory
cyanide
iodoacetamide
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pH 7.5, 40% inhibition at 1 mM
NADP+
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inhibits NADPH oxidation, non-competitive with respect to nitrate
NADPH
NH4+
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inhibits the enzyme under aerobic conditions
nitrite
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competitive with respect to nitrate and non-competitive to NADPH
o-phenanthroline
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p-chloromercuribenzoate
p-hydroxymercuribenzoate
phenanthroline hydrate
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pH 7.5, 1:10, 20% inhibition at 0.1 mM
Phenylglyoxal
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in 0.1 M phosphate, pH 7.3, 4 mM, inactivation after 15 min to 40% and to 20% after 60 min
potassium chlorate
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pH 7.5, slight, 20% inhibition at 5 mM
potassium ethyl xanthate
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Sodium nitrite
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pH 7.5, slight, 50% inhibition at 5 mM
Thiourea
-
-
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FAD
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activates 10fold by addition of 0.004 mM, stabilizes against heat inactivation
NH4+
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slight activation of the enzyme under anaerobic conditions
o-phenanthroline
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40% increase of activity at 2.5 mM
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.024 - 0.029
cytochrome c
2.5
FADH2
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+ nitrate
3
FMNH2
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+ nitrate
0.4 - 3
NADH
0.009 - 4.2
NADPH
0.012 - 0.29
nitrate
additional information
additional information
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Michaelis-Menten kinetics, holo-enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
618
ferricyanide
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275 - 1700
NADH
18 - 1400
NADPH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.04 - 0.065
NADP+
0.15 - 0.18
nitrite
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.003
ectomycchorizae
0.004 - 0.074
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various temperature-sensitive mutants
0.012
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wild-type enzyme, anaerobic conditions, presence of ammonium
0.013
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wild-type enzyme, aerobic conditions, presence of nitrate
0.018
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wild-type enzyme, anaerobic conditions, presence of nitrate
0.019
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wild-type enzyme, anaerobic conditions, presence of nitrate and ammonium
0.02
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activity under aerobic conditions in presence of NH4+
0.063
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nit-1 mutant
0.074
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wild type
0.1
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activity under aerobic conditions
0.3
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activity under anaerobic conditions
0.37
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activity under anaerobic conditions in presence of NH4+
4 - 16.1
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at different stages of purification
4.32
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purified recombinant holo-enzyme, substrate nitrate, pH 7.5, temperature not specified in the publication
177.1
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purified native enzyme, substrate nitrate, pH 7.2, 37C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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wild type after his-tag is removed
7.3
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assay at
7.7
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assay at
additional information
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optima of associated activities
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23 - 28
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assay at
additional information
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a growth temperature above 20C is required
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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free-living cells, yeast extract/mannitol medium
Manually annotated by BRENDA team
non-inoculated
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
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2 * 59000 + 2 * 38000, SDS-PAGE, after heat-treatment all polypeptide chains are 59 kDa
59000
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2 * 59000 + 2 * 38000, SDS-PAGE, after heat-treatment all polypeptide chains are 59 kDa
91000
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2 * 91000, homodimer, SDS-PAGE, smaller bands are caused by proteolytic cleavage
102000
2 * 102000
115000
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1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
116000
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4 * 116000
130000
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1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
132000
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2 * 97000: nit-3 enzyme, 2 * 132000, wild-type
145000
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monodimer, 2 * 145000
150000
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1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
180000 - 200000
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sucrose density gradient, gel-filtration
180000
197000
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gel filtration
204000
214000
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gel filtration
230000
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sucrose density gradient, gel filtration
235000
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sucrose density gradient centrifugation, gel filtration
272000
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wild-type, gel filtration
450000
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native gel electrophoresis
additional information
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gel filtration and analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotrimer
homodimer
multimer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
vapor diffusion against a reservoir of solution 7 from the Hampton Crystal Screen II
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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purified native nitrate reductase, maintaining 50% of its original activity
741637
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
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half-life: 36 h
20 - 60
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purified native nitrate reductase, pH 7.2, maintaining 50% of its original activity
30
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wild-type half-life: 66 min
35
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wild-type half-life: 18.8 min, various temperature sensitive mutant strains: 3.8-18.7 min
47
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half-life: 30 sec
49
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50% loss of activity after 1.5 min
50
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fraction V, pH 7.0, loses all of its activity in 5 min
60
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labile, loss of acitvity after 2 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glutathione stabilizes the enzyme at 1 mM, cysteine as well but not as effective
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loss of activity with ammonium chloride or ammonium nitrate at 10 mM to growing cells, faster decay in washed and resuspended cells
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ethanol
Glycerol
enzyme activity is strongly negatively impacted by increase in solution viscosity using 50% glycerol
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10C, 0.1 M sodium phosphate buffer, pH 7.3, 0.17 M NaCl, 1 mM dithiothreitol, 5 m M EDTA, 0.5 mM PMSF, 1%ethanol, 30% glycerol
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-10C, 50 mM sodium phosphate, pH 6.9, 30% glycerol, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonylfluoride, 0.1 mM FAD, stable for at least 6 months
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-15C, fraction II, most stable, optimal pH: 7.0, 10-20% loss of activity after a month
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-15C, fraction IV, one week, loses about half of its activity
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-15C, fraction V, quite unstable, overnight, loses at least half of its activity
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-20C, stable for 6 months or longer, 5% loss of activity
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-80C, 0.02 mM FAD, several months with only slight loss of the activity
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-80C, 25 mM Mops, 0.1 mM EDTA, pH 7.2
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-80C, extract, 6 months, without loss of activity
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4C or -15C, fraction III, overnight, 50% loss of activity
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4C, half life of 3-5 days
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5C, urea-treated, 0.1 M potassium phosphate buffer, pH 7.3, 5 mM EDTA, 2 mM dithioerythritol, 0.5 mg/ml or more bovine serum albumin, stable for 18 h without loss of activity
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half life of about 2 h with ammonium sulfate or ammonium chloride as sole N-source
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium slufate precipitation, ion-exchange, gel filtration, hydroxylapatite column. FAD and EDTA essential in all buffers during purification
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ammonium sulfate precipitation
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gel filtration
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immobilized metal affinity chromatography and SourceQ15 column anion exchange chromatography
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ion-exchange, ammonium sulfate precipitation, gel filtration
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metal-chelate affinity chromatography for his-tagged proteins, ammonium sulfate fractionation, affinity chromatography
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native enzyme 70fold by ultrafiltration, anion exchange chromatography, and gel filtration
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partially purification of wild-type and nit-3 mutant by streptomycin sulfate precipitation and ammonium sulfate fractionation, of nit-1 mutant by protamine sulfate fractionation, ammonium sulfate precipitation and gel filtration
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partially purified
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protamine sulfate and ammonium sulfate precipitation, hemoglobin-Sepharose column, Bio-Gel A, FAD-Sepharose affinity column. proteolysis during extensive purification
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quick purification by immunoprecipitation with monospecific anti-nitrate reductase serum, stabilization during purification and protection of proteolytic cleavage by addition of phenylmethylsulphonyl fluoride
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recombinant enzyme from Escherichia coli strains TP1000, RK5206, and RK5204 by a two-step affinity purification
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salt fractionation, gel filtration , ion-exchange
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streptomycin sulfate and ammonium sulfate precipitation, ion-exchange, gel filtration, isoelectric focusing, FAD-affinity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of a fusion gene comprising gene the promotor of gene niaD and the Escherichia coli beta-glucuronidase gene, i.e. GUS, for analysis of promotor activity in Aspergillus nidulans
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expressed in Pichia pastoris
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gene nar1, DNA and amino acid sequence determination and analysis, expression study, gene nar1 is part of the nitrate assimilation gene cluster together with genes encoding a high-affinity nitrate transporter and a nitrite reductase, the intergenic region could also act as bidirectional promotor, organisation of coding sequences
gene tbnr1, DNA and amino acid sequence determination and analysis, expression analysis
gene tpnr1, DNA sequence determination and analysis, construction of a cDNA library
sequence comparisons, recombinant enzyme expression of wild-type and mutant enzymes in Escherichia coli strains TP1000, RK5206, and RK5204
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wild-type and mutants are expressed in Escherichia coli JM109(DE3)pLysS
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G811V
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site-directed mutagenesis, an FAD-binding mutant
H654A/H677A
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site-directed mutagenesis, CD spectroscopy shows no negative effects of the introduced mutations on protein secondary structure in comparison to the wild-type protein, but the mutant contains no heme, while the FAD binding ability is not significantly disturbed
R778E
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site-directed mutagenesis, an FAD-binding mutant. The mutant binds essentially the same amount of Moco as does the wild type protein
R921S
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little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity
R921T
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little impact on NADPH and NADH activity, no importance for pyridine nucleotide specificity
R932Q
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1/4 wild type NADPH activity is retained, twice as much NADH activity is present as compared to wild type
R932S
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1/10 wild type NADPH activity is retained, 2/3 of wild type NADH activity
S920D
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important for the enzyme's interaction with the pyridine nucleotide substrates. Mutant retains ~2% of the NADPH activity of the wild type while it has an increased NADH activity, ~15% higher. It is concluded that Ser920 is a ligand involved in binding the 2' phosphate of NADPH in the wild type enzyme
S920D/R932S
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greatest decrease in NADPH activity of all created mutants, shows that Arg932 is a residue interacting with the pyridine nucleotide coenzyme electron donors and that Ser920 and Arg932 have effects on substrate binding and catalytic activity. Both residues may be ligands to the 2' phosphate of NADPH in the wild type cyt b reductase fragment of nitrate reductase
Y780A
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site-directed mutagenesis, an FAD-binding mutant
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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sequential injection analysis flow system for determination of nitrites and nitrates in human serum
medicine
synthesis