Information on EC 1.14.14.47 - nitric-oxide synthase (flavodoxin)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.14.47
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RECOMMENDED NAME
GeneOntology No.
nitric-oxide synthase (flavodoxin)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 L-arginine + 2 reduced flavodoxin + 2 O2 = 2 Nomega-hydroxy-L-arginine + 2 oxidized flavodoxin + 2 H2O
show the reaction diagram
(1a)
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2 L-arginine + 3 reduced flavodoxin + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 oxidized flavodoxin + 4 H2O
show the reaction diagram
overall reaction
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2 Nomega-hydroxy-L-arginine + reduced flavodoxin + 2 O2 = 2 L-citrulline + 2 nitric oxide + oxidized flavodoxin + 2 H2O
show the reaction diagram
(1b)
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine biosynthesis
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Arginine and proline metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
L-arginine,reduced-flavodoxin:oxygen oxidoreductase (nitric-oxide-forming)
Binds heme (iron protoporphyrin IX) and tetrahydrobiopterin. The enzyme, found in bacteria and archaea, consist of only an oxygenase domain and functions together with bacterial ferredoxins or flavodoxins. The orthologous enzymes from plants and animals also contain a reductase domain and use only NADPH as the electron donor (cf. EC 1.14.13.39).
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 L-arginine + 2 reduced flavodoxin + 2 O2
2 Nomega-hydroxy-L-arginine + 2 oxidized flavodoxin + 2 H2O
show the reaction diagram
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-
-
?
2 L-arginine + 2 reduced tetrahydrobiopterin + 2 O2
2 Nomega-hydroxy-L-arginine + 2 oxidized tetrahydrobiopterin + 2 H2O
show the reaction diagram
2 L-arginine + 3 reduced flavodoxin + 4 O2
2 L-citrulline + 2 nitric oxide + 3 oxidized flavodoxin + 4 H2O
show the reaction diagram
2 L-arginine + 3 reduced tetrahydrobiopterin + 4 O2
2 L-citrulline + 2 nitric oxide + 3 oxidized tetrahydrobiopterin + 4 H2O
show the reaction diagram
2 L-arginine + 3 reduced tetrahydrofolate + 4 O2
2 L-citrulline + 2 nitric oxide + 3 oxidized tetrahydrofolate + 4 H2O
show the reaction diagram
2 Nomega-hydroxy-L-arginine + reduced flavodoxin + 2 O2
2 L-citrulline + 2 nitric oxide + oxidized flavodoxin + 2 H2O
show the reaction diagram
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in single turnover experiments with Nomega-hydroxy-L-arginine, NO forms only in the presence of (6R)-tetrahydro-L-biopterin
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?
2 Nomega-hydroxy-L-arginine + reduced tetrahydrobiopterin + 2 O2
2 L-citrulline + 2 nitric oxide + oxidized tetrahydrobiopterin + 2 H2O
show the reaction diagram
N-omega-hydroxy-L-arginine + reduced flavodoxin + 2 O2
2 L-citrulline + 2 nitric oxide + oxidized flavodoxin + 2 H2O
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin
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iron-sulfur centre
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the enzyme's reductase domain utilizes a 2Fe2S cluster for electron transfer
NADH
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small preference for NADH over NADPH
NADPH
tetrahydrobiopterin
tetrahydrofolate
additional information
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enzyme is able to use several different redox partners
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-bromo-7-nitroindazole
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3-[2,4-di(6-amino-4-methylpyridin-2-yl)ethyl]benzonitrile
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inhibitor binds to heme propionate A through a bifurcated H-bond and a pi-pi stacking interaction between the conserved Tyr and aminopyridine group
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4-methylquinolin-2-amine
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among the most potent aminoquinoline inhibitors tested, KS value 0.00080 mM
6,6'-[(5-amino-1,3-phenylene)di(ethane-2,1-diyl)]bis(4-methylpyridin-2-amine)
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compound interacts with the active site Glu243 and heme propionate D through a series of hydrogen bonds between the aminopyridine functional groups. Comparison with inhibition of mammalian NOS isoforms
6,6'-[[(2S,3S)-2-aminobutane-1,3-diyl]bis(oxymethanediyl)]bis(4-methylpyridin-2-amine)
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compound impedes the growth of Bacillus subtilis under oxidative stress an is able to displace the tetrahydrobiopterin cofactor in the Bacillus subtilis enzyme but not in the mouse enzyme
6,6'-[[5-(aminomethyl)-1,3-phenylene]di(ethane-2,1-diyl)]bis(4-methylpyridin-2-amine)
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compound interacts with the active site Glu243 and heme propionate D through a series of hydrogen bonds between the aminopyridine functional groups. Comparison with inhibition of mammalian NOS isoforms
6-([(3R,5S)-5-][[[[(6-amino-4-methylpyridin-2-yl)methoxy]methyl]pyrrolidin-3-yl]oxy]methyl)-4-methylpyridin-2-amine
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compound impedes the growth of Bacillus subtilis under oxidative stress
6-[5-([4-[(6-amino-4-methylpyridin-2-yl)methyl]pyrrolidin-3-yl]oxy)pentyl]-4-methylpyridin-2-amine
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binding is stabilized by a 3.2 A H-bond between the pyrrolidine ring and the carbonyl group of tetrahydrobiopterin
7-nitroindazole
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N,N'-[[(2S)-3-aminopropane-1,2-diyl]bis(oxymethylene-3,1-phenylene)]di(thiophene-2-carboximidamide)
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inhibitor binds by extending outside the active site to interact with a surface adjacent to residue Y357
N-omega-nitro-L-arginine
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N1-[6-[2-(6-amino-4-methylpyridin-2-yl)ethyl]pyridin-2-yl]-N1,N2-dimethylethane-1,2-diamine
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inhibitor binding distorts the pterin binding site by inducing an alternative rotameric position in residue W329. KS value 18.3 microM
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Ngamma-nitro-L-arginine
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competitive
quinolin-2-amine
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among the most potent aminoquinoline inhibitors tested, KS value 0.00125 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
tryptophan
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stimulates oxidation of substrate L-arginine. Tryptophan or a derivative thereof may bind in the enzyme's pterin site, participate in arginine oxidation, and become nitrated at the 4-position
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0082
L-arginine
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pH 7.5, 25°C
0.0001
reduced tetrahydrobiopterin
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pH 7.6, 25°C
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0.0004
reduced tetrahydrofolate
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pH 7.6, 25°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.183
reduced tetrahydrobiopterin
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pH 7.6, 25°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0146
Ngamma-nitro-L-arginine
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pH 7.5, 25°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0366 - 0.0533
4-methylquinolin-2-amine
0.0027
7-nitroindazole
Bacillus subtilis;
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chimera between enzyme and flavodoxin YkuN, pH 7.4, 35°C
0.0102
N-omega-nitro-L-arginine
Bacillus subtilis;
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chimera between enzyme and flavodoxin YkuN, pH 7.4, 35°C
0.0273 - 0.0639
quinolin-2-amine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
318
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pH 7.5, 25°C
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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Manually annotated by BRENDA team
PDB
SCOP
CATH
UNIPROT
ORGANISM
Bacillus subtilis (strain 168);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
86000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
2 * 43000, calculated
additional information
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enzyme contains a catalytic oxygenase domain with a fused reductase domain. The reductase domain utilizes a 2Fe2S cluster for electron transfer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
binding of substrate L-arginine or cofactor tetrahydrobiopterin converts nitric oxide synthase from a loose dimer, with an exposed active center and higher sensitivity to proteolysis, to a tight dimer competent for catalysis
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in complex with a number of inhibitors
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in complex with cofactor tetrahydrofolate and substrate L-arginine or the intermediate Nomega-hydroxy-L-arginine to 1.9 or 2.2 Å resolution, respectively
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in complex with inhibitors that target both the active and pterin sites
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incomplex with inhibitors 6-([(3R,5S)-5-][[[[(6-amino-4-methylpyridin-2-yl)methoxy]methyl]pyrrolidin-3-yl]oxy]methyl)-4-methylpyridin-2-amine and 6,6'-[[(2S,3S)-2-aminobutane-1,3-diyl]bis(oxymethanediyl)]bis(4-methylpyridin-2-amine)
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structures of the Fe(III)-NO complex with Nomega-hydroxy-L-arginine show a nearly linear nitrosyl group, and in one subunit, partial nitrosation of bound Nomega-hydroxy-L-arginine. In the Fe(II)-NO complexes, the protonated Nomega-hydroxy-L-arginine Nomega atom forms a short hydrogen bond with the heme-coordinated NO nitrogen, but active-site water molecules are out of hydrogen bonding range with the distal NO oxygen. The L-Arg guanidinium interacts more weakly and equally with both NO atoms, and an active-site water molecule hydrogen bonds to the distal NO oxygen
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wild-tye and mutant I208V, in complex with inhibitors
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wild-type in complex with inhibitors N-omega-nitro-L-arginine and 3-bromo-7-nitroindazole
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to 3.2 A resolution. Residue Lys-356 (Bacillus subtilis NOS) is changed to Arg-365 (gsNOS), substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme
enzyme exhibits two conformations in the absence of substrate. The addition of L-Arg stabilizes the conformer more similar to the Mus muculus enzyme, whereas N-hydroxy-L-arginine stabilizes the conformer more similar to the Bacillus subtilis enzyme, although both substrates introduce a positive electrostatic potential to the distal heme pocket
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to 2.4 A resolution. Heme and inhibitor S-ethylisothiourea are bound at the active site, while the intersubunit site has NAD+ bound. Enzyme is a dimer, with NAD+ in the interface ligand binding site. Heme is buried in the interior of each monomer
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
melting temperature
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E25A/E26A/E316A
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mutant facilitates crystallization
E25A/E26A/E316A/Y357F
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mutant facilitates crystallization
I208V
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crystallization and inhibition data
P332G
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mutation at the center of the dimer interface, mutant displays significantly more monomer content than wild-type
P332G/A333S
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mutation at the center of the dimer interface, both mutations are necessary to mimic interactions at the dimer interface displayed by the mouse enzyme
W66L
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
W66Y
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
E25A/E26A/E316A
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mutant facilitates crystallization
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E25A/E26A/E316A/Y357F
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mutant facilitates crystallization
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I208V
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crystallization and inhibition data
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P332G
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mutation at the center of the dimer interface, mutant displays significantly more monomer content than wild-type
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P332G/A333S
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mutation at the center of the dimer interface, both mutations are necessary to mimic interactions at the dimer interface displayed by the mouse enzyme
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W66F
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
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W66H
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
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W66L
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
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W66Y
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mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
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L356R
mutation Lys-356 (Bacillus subtilis NOS) to Arg-365 (gsNOS) substitution alters the conformation of a conserved Asp carboxylate, resulting in movement of an Ile residue toward the heme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
Show AA Sequence (1417 entries)
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