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Information on EC 1.14.14.47 - nitric-oxide synthase (flavodoxin) and Organism(s) Bacillus subtilis and UniProt Accession O05268

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IUBMB Comments
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).
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Bacillus subtilis
UNIPROT: O05268
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Word Map
The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
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Synonyms
sanos, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
nitric oxide synthetase
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NO synthase
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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).
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 tetrahydrobiopterin + 2 O2
2 Nomega-hydroxy-L-arginine + 2 oxidized tetrahydrobiopterin + 2 H2O
show the reaction diagram
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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
2 L-arginine + 3 tetrahydrobiopterin + 4 O2
2 L-citrulline + 2 nitric oxide + 3 oxidized tetrahydrobiopterin + 4 H2O
show the reaction diagram
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overall reaction
-
?
2 L-arginine + 3 tetrahydrofolate + 4 O2
2 L-citrulline + 2 nitric oxide + 3 oxidized tetrahydrofolate + 4 H2O
show the reaction diagram
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overall reaction
-
?
2 Nomega-hydroxy-L-arginine + tetrahydrobiopterin + 2 O2
2 L-citrulline + 2 nitric oxide + oxidized tetrahydrobiopterin + 2 H2O
show the reaction diagram
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-
-
?
N-omega-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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-
?
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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tetrahydrobiopterin
NO formation requires a bound tetrahydropteridine, and the kinetic effects are consistent with it donating an electron to the Fe(II)O2 intermediate during the reaction
tetrahydrofolate
additional information
-
enzyme is able to use several different redox partners
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
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
inhibitor binds to heme propionate A through a bifurcated H-bond and a pi-pi stacking interaction between the conserved Tyr and aminopyridine group
4-methylquinolin-2-amine
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)
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)
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)
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
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
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)
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
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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quinolin-2-amine
among the most potent aminoquinoline inhibitors tested, KS value 0.00125 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0001
tetrahydrobiopterin
pH 7.6, 25°C
0.0004
tetrahydrofolate
pH 7.6, 25°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.183
tetrahydrobiopterin
pH 7.6, 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
chimera between enzyme and flavodoxin YkuN, pH 7.4, 35°C
0.0102
N-omega-nitro-L-arginine
Bacillus subtilis
chimera between enzyme and flavodoxin YkuN, pH 7.4, 35°C
0.0273 - 0.0639
quinolin-2-amine
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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enzyme activity is important for maintaining normal growth at the log-to-stationary transition phase. Enzyme produces NO in living cells and uses available cellular redox partners that are not normally committed to NO production. The promiscuous bacterial reductase also supports NO synthesis by the oxygenase domain of mammalian nitric oxide synthase expressed in Escherichia coli
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
in complex with a number of inhibitors
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
in complex with inhibitors that target both the active and pterin sites
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)
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
wild-tye and mutant I208V, in complex with inhibitors
wild-type in complex with inhibitors N-omega-nitro-L-arginine and 3-bromo-7-nitroindazole
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E25A/E26A/E316A
mutant facilitates crystallization
E25A/E26A/E316A/Y357F
mutant facilitates crystallization
I208V
crystallization and inhibition data
P332G
mutation at the center of the dimer interface, mutant displays significantly more monomer content than wild-type
P332G/A333S
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
mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
W66Y
mutation modulates hydrogen bond interaction to the thiolate ligand which controls the stability of various NOS intermediates
additional information
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
development of a robust enzyme assay for measuring NOS activity and inhibition
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Wang, Z.; Lawson, R.J.; Buddha, M.R.; Wei, C.; Crane, B.R.; Munro, A.W.; Stuehr, D.J.
Bacterial flavodoxins support nitric oxide production by Bacillus subtilis nitric-oxide synthase
J. Biol. Chem.
282
2196-2202
2007
Bacillus subtilis
Manually annotated by BRENDA team
Gusarov, I.; Starodubtseva, M.; Wang, Z.Q.; McQuade, L.; Lippard, S.J.; Stuehr, D.J.; Nudler, E.
Bacterial nitric-oxide synthases operate without a dedicated redox partner
J. Biol. Chem.
283
13140-13147
2008
Bacillus anthracis, Bacillus subtilis
Manually annotated by BRENDA team
Hannibal, L.; Somasundaram, R.; Tejero, J.; Wilson, A.; Stuehr, D.
Influence of heme-thiolate in shaping the catalytic properties of a bacterial nitric-oxide synthase
J. Biol. Chem.
286
39224-39235
2011
Bacillus subtilis
Manually annotated by BRENDA team
Pant, K.; Bilwes, A.M.; Adak, S.; Stuehr, D.J.; Crane, B.R.
Structure of a nitric oxide synthase heme protein from Bacillus subtilis
Biochemistry
41
11071-11079
2002
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Santolini, J.; Roman, M.; Stuehr, D.; Mattioli, T.
Resonance Raman study of Bacillus subtilis NO synthase-like protein: Similarities and differences with mammalian NO synthases
Biochemistry
45
1480-1489
2006
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Pant, K.; Crane, B.R.
Nitrosyl-heme structures of Bacillus subtilis nitric oxide synthase have implications for understanding substrate oxidation
Biochemistry
45
2537-2544
2006
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Holden, J.K.; Dejam, D.; Lewis, M.C.; Huang, H.; Kang, S.; Jing, Q.; Xue, F.; Silverman, R.B.; Poulos, T.L.
Inhibitor bound crystal structures of bacterial nitric oxide synthase
Biochemistry
54
4075-4082
2015
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Holden, J.K.; Lewis, M.C.; Cinelli, M.A.; Abdullatif, Z.; Pensa, A.V.; Silverman, R.B.; Poulos, T.L.
Targeting bacterial nitric oxide synthase with aminoquinoline-based inhibitors
Biochemistry
55
5587-5594
2016
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Adak, S.; Aulak, K.S.; Stuehr, D.J.
Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis
J. Biol. Chem.
277
16167-16171
2002
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Brunel, A.; Wilson, A.; Henry, L.; Dorlet, P.; Santolini, J.
The proximal hydrogen bond network modulates Bacillus subtilis nitric-oxide synthase electronic and structural properties
J. Biol. Chem.
286
11997-12005
2011
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Holden, J.K.; Lim, N.; Poulos, T.L.
Identification of redox partners and development of a novel chimeric bacterial nitric oxide synthase for structure activity analyses
J. Biol. Chem.
289
29437-29445
2014
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Holden, J.K.; Kang, S.; Hollingsworth, S.A.; Li, H.; Lim, N.; Chen, S.; Huang, H.; Xue, F.; Tang, W.; Silverman, R.B.; Poulos, T.L.
Structure-based design of bacterial nitric oxide synthase inhibitors
J. Med. Chem.
58
994-1004
2015
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Pant, K.; Crane, B.R.
Structure of a loose dimer: an intermediate in nitric oxide synthase assembly
J. Mol. Biol.
352
932-940
2005
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team
Holden, J.K.; Li, H.; Jing, Q.; Kang, S.; Richo, J.; Silverman, R.B.; Poulos, T.L.
Structural and biological studies on bacterial nitric oxide synthase inhibitors
Proc. Natl. Acad. Sci. USA
110
18127-18131
2013
Bacillus subtilis (O34453), Bacillus subtilis 168 (O34453)
Manually annotated by BRENDA team