Information on EC 1.14.13.165 - nitric-oxide synthase [NAD(P)H-dependent]

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
1.14.13.165
-
RECOMMENDED NAME
GeneOntology No.
nitric-oxide synthase [NAD(P)H-dependent]
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2 L-arginine + 2 NAD(P)H + 2 H+ + 2 O2 = 2 Nomega-hydroxy-L-arginine + 2 NAD(P)+ + 2 H2O
show the reaction diagram
(1a)
-
-
-
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
overall reaction
-
-
-
2 Nomega-hydroxy-L-arginine + NAD(P)H + H+ + 2 O2 = 2 L-citrulline + 2 nitric oxide + NAD(P)+ + 2 H2O
show the reaction diagram
(1b)
-
-
-
SYSTEMATIC NAME
IUBMB Comments
L-arginine,NAD(P)H:oxygen oxidoreductase (nitric-oxide-forming)
Binds heme (iron protoporphyrin IX) and tetrahydrobiopterin. Most of the bacterial and archaeal enzymes consist of only an oxidase domain and function together with bacterial ferredoxins [1-2]. The enzyme from the delta-proteobacterium Sorangium cellulosum also includes a reductase domain that binds FAD, FMN and a [2Fe-2S] cluster [3]. The similar enzymes from plants and animals use only NADPH as acceptor (cf. EC 1.14.13.39).
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
nitric oxide synthetase
-
-
-
-
NO synthase
-
-
-
-
SANOS
P0A004
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
methicillin-resistant strain
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
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
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 L-arginine + 2 NAD(P)H + 2 H+ + 2 O2
2 Nomega-hydroxy-L-arginine + 2 NAD(P)+ + 2 H2O
show the reaction diagram
-
-
1st step of reaction
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
-
-
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
-
overall reaction
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
-
flavodoxins YkuN and YkuP as well as protein cisJ may be used as redox partners, enzyme uses different available cellular redox partner to support its NO synthesis
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
-
overall reaction. Flavodoxins YkuN and YkuP support catalysis as kinetically competent redox partners. When an NADPH-utilizing bacterial flavodoxin reductase is added to reduce YkuP or YkuN, both support nitric oxide synthesis from either L-arginine or N-hydroxyarginine, with YkuN being more efficient
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
NADPH may be substituted by flavoproteins or tetrahydrofolate
-
-
?
2 L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
tetrahydrobiopterin or tetrahydrofolate may act as redox partners
overall reaction
-
?
2 Nomega-hydroxy-L-arginine + 3 NAD(P)H + 3 H+ + 4 O2
2 L-citrullin + 2 nitric oxide + 3 NAD(P)+ + 4 H2O
show the reaction diagram
-
-
overall reaction. Flavodoxins YkuN and YkuP support catalysis as kinetically competent redox partners. When an NADPH-utilizing bacterial flavodoxin reductase is added to reduce YkuP or YkuN, both support nitric oxide synthesis from either L-arginine or N-hydroxyarginine, with YkuN being more efficient
-
?
2 Nomega-hydroxy-L-arginine + NAD(P)H + H+ + 2 O2
2 L-citrulline + 2 nitric oxide + NAD(P)+ + 2 H2O
show the reaction diagram
-
-
2nd step of reaction
-
?
additional information
?
-
-
the complex between enzyme and the unusual tryptophanyl-tRNA synthetase TrpRS II catalyzes the regioselective nitration of tryptophan at the 4-position. The enzyme alone will catalyze 4-nitrotryptophan production, but yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-tryptophan formation exhibits saturation behavior with tryptophan and is completely inhibited by the addition of the mammalian nitric-oxide synthase cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(6R)-L-erythro-5,6,7,8-tetrahydrobiopterin
-
-
NADH
-
small preference for NADH over NADPH
NADPH
-
small preference for NADH over NADPH
tetrahydrobiopterin
-
-
tetrahydrobiopterin
-
or tetrahydrofolate
tetrahydrofolate
-
-
tetrahydrofolate
-
or tetrahydrobiopterin
iron-sulfur centre
-
the enzyme's reductase domain utilizes a 2Fe2S cluster for electron transfer
additional information
-
enzyme is able to use several different redox partners
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Iron
-
in the presence of L-arginine and tetrahydrofolate, wild-type and mutants W66F and W66H exist in the typical Fe(III) high-spin configuration
Iron
-
the enzyme's reductase domain utilizes a 2Fe2S cluster for electron transfer
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Ngamma-nitro-L-arginine
-
competitive
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
tryptophan
-
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0082
-
L-arginine
-
pH 7.5, 25C
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0146
-
Ngamma-nitro-L-arginine
-
pH 7.5, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
318
-
-
pH 7.5, 25C
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
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 ACCESSION NO.
LITERATURE
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
P0A004, -
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
partial
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
P0A004, -
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
W66F
-
mutation changes midpoint potential from -361 mV for wild-type to -427 mV. Mutant displays 2.5fold lower activity when reaction is supported by flavoproteins or NADPH instead of tetrahydrofolate
W66H
-
mutation changes midpoint potential from -361 mV for wild-type to -302 mV. Activity is similar to wild-type