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Information on EC 1.7.1.3 - nitrate reductase (NADPH) and Organism(s) Neurospora crassa and UniProt Accession P08619
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1.7.1.3 nitrate reductase (NADPH)IUBMB Comments
An iron-sulfur molybdenum flavoprotein.
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Word Map
- 1.7.1.3
- neurospora
- crassa
- nidulans
- molybdenum
-
molybdenum-containing
-
nadph-cytochrome
- molybdopterin
-
positive-acting
-
pathway-specific
-
viologen-nitrate
-
reductase-deficient
-
nitrogen-related
- tungstate
-
nadh:nitrate
- synthesis
- analysis
- medicine
The taxonomic range for the selected organisms is: Neurospora crassa
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Synonyms
nit-3, nadph-nitrate reductase, nadph-dependent nitrate reductase, nadph:nr, assimilatory nadph-nitrate reductase, nadph:nitrate reductase, nitrate reductase (nadph), 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
assimilatory NADPH-nitrate reductase
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-
-
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Assimilatory nitrate reductase
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-
-
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assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase
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-
-
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NADPH-dependent nitrate reductase
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-
-
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NADPH-nitrate reductase
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-
-
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NADPH2:nitrate oxidoreductase
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-
-
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NADPH:nitrate reductase
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-
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nitrate reductase (NADPH)
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-
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nitrate reductase (reduced nicotinamide adenine dinucleotide phosphate)
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-
-
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triphosphopyridine nucleotide-nitrate reductase
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-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nitrite + NADP+ + H2O = nitrate + NADPH + H+
multicenter redox enzyme. Ser920, Arg921 and Arg932 are suggested to be the key enzymes to investigate for a role in determining pyridine nucleotide specificity. Arg932 may be playing a role in binding the adenine ring of NADPH
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nitrite + NADP+ + H2O = nitrate + NADPH + H+
sulfhydryl groups may participate in the binding of the protein subunits
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
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oxidation
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-
-
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reduction
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-
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SYSTEMATIC NAME
IUBMB Comments
nitrite:NADP+ oxidoreductase
An iron-sulfur molybdenum flavoprotein.
CAS REGISTRY NUMBER
COMMENTARY
9029-28-1
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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
nitrate + reduced anthraquinone 2-sulfonate
nitrite + anthraquinone 2-sulfonate
-
-
-
-
?
additional information
?
-
-
NADPH-dependent cytochrome c reducing activity by the holo-enzyme is determined with FAD and NADPH spectroscopically at 550 nm and 340 nm. Apo-nitrate reductase has a marginally lower, about 10% reduced cytochrome c reducing activity, which correlates to its 15% reduced heme content
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-
?
nitrate + NADPH
nitrite + NADP+ + H2O
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20-fold higher activity with NADPH than with NADH
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ir
nitrate + NADPH
nitrite + NADP+ + H2O
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4 activities: NADPH-nitrate reductase, FADH-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
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4 activities: NADPH-nitrate reductase, FADH-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
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4 activities: NADPH-nitrate reductase, FADH-nitrate reductase, reduced methyl viologen-nitrate reductase and NADPH-cytochrome c reductase
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
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associated FAD-nitrate reductase and methylviologen-nitrate activity
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?
nitrate + NADPH
nitrite + NADP+ + H2O
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first step in nitrate assimilation
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?
nitrate + NADPH
nitrite + NADP+ + H2O
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key enzyme in the assimilation path of nitrate to ammonium. Crude extracts possess endogenous NADPH regenerating systems capable of providing reducing equivalents for effective nitrate reduction in vitro
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
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first step in the reduction of nitrate to ammonia, biosynthesis of amino acids and other nitrogen-containing cell constituents
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?
NATURAL SUBSTRATE
NATURAL 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
nitrate + NADPH
nitrite + NADP+ + H2O
-
first step in nitrate assimilation
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
-
key enzyme in the assimilation path of nitrate to ammonium. Crude extracts possess endogenous NADPH regenerating systems capable of providing reducing equivalents for effective nitrate reduction in vitro
-
?
nitrate + NADPH
nitrite + NADP+ + H2O
-
first step in the reduction of nitrate to ammonia, biosynthesis of amino acids and other nitrogen-containing cell constituents
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FAD
involved in electron transfer from NADPH to the enzyme molybdenum center where reduction of nitrate to nitrite takes place
heme
involved in electron transfer from NADPH to the enzyme molybdenum center where reduction of nitrate to nitrite takes place
molybdenum cofactor
cofactor is necessary and sufficient to induce dimer formation. The molybdenum center of nitrate reductase reconstituted in vitro from apo-enzyme and cofactor shows an EPR spectrum identical to holo-enzyme. Insertion of this cofactor into the enzyme occurs independent from the insertion of any other NR redox cofactor
molybdenum cofactor
-
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
molybdopterin
-
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
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Molybdenum
Molybdenum
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1 mol per mol enzyme protein, activation of enzyme after urea treatment, little stimulation
Molybdenum
-
a functional nitrate reductase can be generated that lacks both the FAD and heme cofactors leaving the Mo active site as the sole redox active centre
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
additional information
-
protection of inactivation by FAD and restorage by dithiothreitol
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
enzyme can be reactivated by molybdenum and dithioerythritol
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
Michaelis-Menten kinetics, holo-enzyme
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0.25
nitrate
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recombinant holo-enzyme, pH 7.5, temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.32
-
purified recombinant holo-enzyme, substrate nitrate, pH 7.5, temperature not specified in the publication
additional information
additional information
-
one unit is defined as that amount of enzyme which results in the formation of 0.000001 mM of nitrite
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
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catalytic voltammetry of enzyme variants on a modified Au electrode with the electrochemically reduced forms of benzyl viologen and anthraquinone sulfonate as artificial electron donors. The biopolymer chitosan entraps nitrate reductase on the electrode noncovalently. Both an enzyme form lacking the FAD cofactor and one lacking both the FAD and heme cofactors show catalytic nitrate reductase activity, removal of the heme cofactor results in a more significant effect on the rate of nitrate reduction
additional information
-
active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization, molybdenum cofactor-dependent enzyme maturation, overview
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
115000
-
1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
130000
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1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
150000
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1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
in presence of molybdenum cofactor, enzyme forms a dimer, gel filtration, sedimentation velocity analysis
dimer
homodimer
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holo-enzyme, determination with SDS-PAGE, gel filtration and analytical ultracentrifugation. The enzyme has a largely hydrophobic dimer interface
additional information
dimer
-
1 * 130000 + 1 * 115000, SDS-PAGE, homodimer of 2 * 150000 suggested
additional information
-
nit-1 mutant enzyme is the apoprotein of nitrate reductase
additional information
-
nit-1 mutant enzyme is the apoprotein of nitrate reductase
additional information
-
aggregate of two different polypeptide chains: one responsible for transport of electrons fom NADPH to FAD or cytochrome c, nit-1 enzyme. The other transfers electrons from FAD via molybdenum to nitrate, nit-3 enzyme
additional information
-
the apo-enzyme dissociates completely into monomers, the ratio between monomeric and dimeric apo-NR does not change significantly upon a 20fold dilution. Active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization, molybdenum cofactor-dependent enzyme maturation, overview. Enzyme domain structure, overview
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
-
site-directed mutagenesis, an FAD-binding mutant. The mutant binds essentially the same amount of Moco as does the wild type protein
R921S
-
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
-
1/4 wild type NADPH activity is retained, twice as much NADH activity is present as compared to wild type
R932S
-
1/10 wild type NADPH activity is retained, 2/3 of wild type NADH activity
S920D
-
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
-
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
additional information
additional information
-
nit-1 mutant: lacks all activities except FAD-dependent NADPH:cytochrome c reductase activity,nit-2 mutant: reduced FAD:- and reduced methyl viologen:nitrate reductase activities but lacks the other two activities
additional information
-
nit-3 mutant (FGSC 262): reduced FAD-nitrate reductase and reduced methylviologen-nitrate reductase activities
additional information
-
nit-1 mutant, suggested to produce the complete apoenzyme
additional information
-
several mutations of recombinant cyt b reductase fragment of nitrate reductase in the region Ser920, Arg921 and Arg932 are created. Conversion from NADPH-specific to virtually NADH-specific cyt b reductase fragment of nitrate reductase
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40
40
-
less than 50% loss of activity in 60 min for wild type and S920D mutant
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
glutathione stabilizes the enzyme at 1 mM, cysteine as well but not as effective
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10°C, 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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-10°C, 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
-
-15°C, fraction II, most stable, optimal pH: 7.0, 10-20% loss of activity after a month
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-15°C, fraction V, quite unstable, overnight, loses at least half of its activity
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5°C, 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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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
metal-chelate affinity chromatography for his-tagged proteins, ammonium sulfate fractionation, affinity chromatography
-
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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protamine sulfate and ammonium sulfate precipitation, hemoglobin-Sepharose column, Bio-Gel A, FAD-Sepharose affinity column. proteolysis during extensive purification
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recombinant enzyme from Escherichia coli strains TP1000, RK5206, and RK5204 by a two-step affinity purification
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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
sequence comparisons, recombinant enzyme expression of wild-type and mutant enzymes in Escherichia coli strains TP1000, RK5206, and RK5204
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Pan, S.S.; Nason, A.
Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa
Biochim. Biophys. Acta
523
297-313
1978
Neurospora crassa, Neurospora crassa STA4
Nicholas, D.J.D.; Nason, A.
Molybdenum and nitrate reductase. II. Molybdenum as a constituent of nitrate reductase
J. Biol. Chem.
207
353-360
1954
Neurospora crassa
Nason, A.; Evans, H.J.
Triphosphopyridine nucleotide-nitrate reductase in Neurospora
J. Biol. Chem.
202
655-673
1953
Neurospora crassa, Neurospora crassa 5297a
Shiraishi, N.; Croy, C.; Kaur, J.; Campbell, W.H.
Engineering of pyridine nucleotide specificity of nitrate reductase: mutagenesis of recombinant cytochrome b reductase fragment of Neurospora crassa NADPH:nitrate reductase
Arch. Biochem. Biophys.
358
104-115
1998
Neurospora crassa
Savidov, N.A.; Alikulov, Z.A.; Lips, S.H.
Identification of an endogenous NADPH-regenerating system coupled to nitrate reduction in vitro in plant and fungal crude extracts
Plant Sci.
133
33-45
1998
Hordeum vulgare, Neurospora crassa
-
Horner, R.D.
Purification and comparison of nit-1 and wild-type NADPH:nitrate reductases of Neurospora crassa
Biochim. Biophys. Acta
744
7-15
1983
Neurospora crassa
-
Tachiki, T.; Nason, A.
Preparation and proterties of apoenzyme of nitrate reductases from wild-type and nit-3 mutant of Neurospora crassa
Biochim. Biophys. Acta
744
16-22
1983
Neurospora crassa
Antoine, A.D.
Purification and properties of the nitrate reductase isolated from Neurospora crassa mutant nit-3. Kinetics, molecular weight determination, and cytochrome involvement
Biochemistry
13
2289-2294
1974
Neurospora crassa
Ringel, P.; Krausze, J.; Van Heuvel, J.; Curth, U.; Pierik, A.; Herzog, S.; Mendel, R.; Kruse, T.
Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa
J. Biol. Chem.
288
14657-14671
2013
Neurospora crassa (P08619)
Ringel, P.; Krausze, J.; Van Heuvel, J.; Curth, U.; Pierik, A.; Herzog, S.; Mendel, R.; Kruse, T.
Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa
J. Biol. Chem.
288
14657-14671
2013
Neurospora crassa
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