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Information on EC 1.14.13.39 - nitric-oxide synthase (NADPH)

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IUBMB Comments
The enzyme consists of linked oxygenase and reductase domains. The eukaryotic enzyme binds FAD, FMN, heme (iron protoporphyrin IX) and tetrahydrobiopterin, and its two domains are linked via a regulatory calmodulin-binding domain. Upon calcium-induced calmodulin binding, the reductase and oxygenase domains form a complex, allowing electrons to flow from NADPH via FAD and FMN to the active center. The reductase domain of the enzyme from the bacterium Sorangium cellulosum utilizes a [2Fe-2S] cluster to transfer the electrons from NADPH to the active center. cf. EC 1.14.14.47, nitric-oxide synthase (flavodoxin).
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UNIPROT: P29474
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Word Map
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
Synonyms
nos, inducible nitric oxide synthase, endothelial nitric oxide synthase, inducible no synthase, neuronal nitric oxide synthase, inducible nos, endothelial no synthase, endothelial nos, neuronal nos, no-synthase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
endothelial nitric oxide synthase
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endothelial nitric-oxide synthase
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endothelial NOS
nitric oxide synthase
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nitric-oxide synthase
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NO synthase type III
eNOS
endothelium-derived relaxation factor-forming enzyme
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endothelium-derived relaxing factor synthase
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NADPH-diaphorase
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nitric oxide synthase
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nitric oxide synthetase
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NO synthase
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synthetase, nitric oxide
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2 L-arginine + 3 NADPH + 3 H+ + 4 O2 = 2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
show the reaction diagram
interaction and electrontransfer between enzyme domains and bound cofactors calmodulin, FMN, FAD, tetrahydrobiopterin, heme, and NADPH, overview
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
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oxidation
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reduction
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SYSTEMATIC NAME
IUBMB Comments
L-arginine,NADPH:oxygen oxidoreductase (nitric-oxide-forming)
The enzyme consists of linked oxygenase and reductase domains. The eukaryotic enzyme binds FAD, FMN, heme (iron protoporphyrin IX) and tetrahydrobiopterin, and its two domains are linked via a regulatory calmodulin-binding domain. Upon calcium-induced calmodulin binding, the reductase and oxygenase domains form a complex, allowing electrons to flow from NADPH via FAD and FMN to the active center. The reductase domain of the enzyme from the bacterium Sorangium cellulosum utilizes a [2Fe-2S] cluster to transfer the electrons from NADPH to the active center. cf. EC 1.14.14.47, nitric-oxide synthase (flavodoxin).
CAS REGISTRY NUMBER
COMMENTARY hide
125978-95-2
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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 + 3 NADPH + 3 H+ + 4 O2
2 citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
show the reaction diagram
2 L-arginine + 3 NADPH + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
show the reaction diagram
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
show the reaction diagram
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?
additional information
?
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NATURAL SUBSTRATE
NATURAL 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 + 3 NADPH + 3 H+ + 4 O2
2 L-citrulline + 2 nitric oxide + 3 NADP+ + 4 H2O
show the reaction diagram
2 L-arginine + 3 NADPH + 4 O2 + 3 H+
2 L-citrulline + 2 NO + 3 NADP+ + 4 H2O
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(6R)-5,6,7,8-tetrahydro-L-biopterin
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NADPH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+/calmodulin
calmodulin activates electron transfer from NADPH through three reductase domains to the oxygenase domain, controls constitutive isoforms through regulation of electrontransfer between NADPH and heme
Iron
heme iron, FMN/heme electron transfer
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N''-nitroguanidine
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3-[cis-4'-[(6''-aminopyridin-2''-yl)methyl]pyrrolidin-3'-ylamino]propan-1-ol
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4-oxononenal
ONE, a highly bioreactive agent, able to inhibit eNOS activity and NO production. It can posttranslationally modify the enzyme in the placenta
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L-N-methylarginine
NOS inhibitor, complete inhibition at 0.5 mM
L-Nomega-nitroarginine-(4R)-amino-L-proline amide
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L-Nomega-nitroarginine-2,4-L-diaminobutyramide
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L-omega-monomethyl L-arginine
potent competitive eNOS inhibitor, complete inhibition at 10 mM
N1-[cis-4'-[(6''-amino-4''-methylpyridin-2''-yl)methyl]pyrrolidin-3'-yl]-N2-(4'-chlorobenzyl)ethane-1,2-diamine
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N1-[cis-4'-[(6''-aminopyridin-2''-yl)methyl]pyrrolidin-3'-yl]ethane-1,2-diamine
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N1-[trans-4'-[(6''-amino-4''-methylpyridin-2''-yl)methyl]pyrrolidin-3'-yl]-N2-(3'-chlorobenzyl)ethane-1,2-diamine
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nanoshutter NS1
mixture of (2S,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-2-([2-(ethyl(4-[(E)-2-(4-nitrophenyl)ethenyl]phenyl)amino)ethyl]carbamoyl)-4-hydroxyoxolan-3-yl dihydrogen phosphate and (2R,3R,4R,5S)-2-(6-amino-9H-purin-9-yl)-5-([2-(ethyl(4-[(E)-2-(4-nitrophenyl)ethenyl]phenyl)amino)ethyl]carbamoyl)-4-hydroxyoxolan-3-yl dihydrogen phosphate. The NOS inhibitor targets the reductase domain of the enzyme
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Nomega-nitro-L-arginine methylester
L-NAME, the NOS inhibitor targets the oxygenase domain of the enzyme
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2',3'-bis-O-(carboxymethyl)-5'-deoxy-5'-(4-([methyl(4-[(1E,3E)-4-[4-(methylamino)phenyl]buta-1,3-dien-1-yl]phenyl)amino]methyl)-1H-1,2,3-triazol-1-yl)adenosine
nanotrigger NT2-2
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2'-O-(carboxymethyl)-5'-deoxy-5'-(4-([methyl(4-[(1E,3E)-4-[4-(methylamino)phenyl]buta-1,3-dien-1-yl]phenyl)amino]methyl)-1H-1,2,3-triazol-1-yl)adenosine
nanotrigger NT2-6
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3'-O-(carboxymethyl)-5'-deoxy-5'-(4-([methyl(4-[(1E,3E)-4-[4-(methylamino)phenyl]buta-1,3-dien-1-yl]phenyl)amino]methyl)-1H-1,2,3-triazol-1-yl)adenosine
nanotrigger NT2-4
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5'-(4-([(4-[(1E,3E)-4-(4-aminophenyl)buta-1,3-dien-1-yl]phenyl)(methyl)amino]methyl)-1H-1,2,3-triazol-1-yl)-2',3'-bis-O-(carboxymethyl)-5'-deoxyadenosine
nanotrigger NT2-3
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5'-(4-([(4-[(1E,3E)-4-(4-aminophenyl)buta-1,3-dien-1-yl]phenyl)(methyl)amino]methyl)-1H-1,2,3-triazol-1-yl)-2'-O-(carboxymethyl)-5'-deoxyadenosine
nanotrigger NT2-7
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5'-(4-([(4-[(1E,3E)-4-(4-aminophenyl)buta-1,3-dien-1-yl]phenyl)(methyl)amino]methyl)-1H-1,2,3-triazol-1-yl)-3'-O-(carboxymethyl)-5'-deoxyadenosine
nanotrigger NT2-5
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5'-(4-([(4-[(1E,3E)-4-(4-aminophenyl)buta-1,3-dien-1-yl]phenyl)(methyl)amino]methyl)-1H-1,2,3-triazol-1-yl)-5'-deoxyadenosine
nanotrigger NT2-9
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5'-[2-[ethyl[4-[4-(4-aminophenyl)-1,3-butadienyl]phenyl]amino]ethylamino]-5'-oxo-5'-deoxyadenosine 2'-phosphoric acid
nanotrigger NT1
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Ca2+/calmodulin
calmodulin activates electron transfer from NADPH through three reductase domains to the oxygenase domain, controls constitutive isoforms through regulation of electrontransfer between NADPH and heme
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Calmodulin
additional information
design and synthesis of a series of two-photon absorbing and photoactivatable NADPH analogues (NT 1 and NT2). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. Two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation is observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells, structure-activity relationship of NTs in the cell context, overview
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
(4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N''-nitroguanidine
wild type eNOS
0.3666
3-[cis-4'-[(6''-aminopyridin-2''-yl)methyl]pyrrolidin-3'-ylamino]propan-1-ol
wild type eNOS
0.11
L-Nomega-nitroarginine-(4R)-amino-L-proline amide
wild type eNOS
0.107
L-Nomega-nitroarginine-2,4-L-diaminobutyramide
wild type eNOS
0.0852
N1-[cis-4'-[(6''-amino-4''-methylpyridin-2''-yl)methyl]pyrrolidin-3'-yl]-N2-(4'-chlorobenzyl)ethane-1,2-diamine
wild type eNOS
0.4167
N1-[cis-4'-[(6''-aminopyridin-2''-yl)methyl]pyrrolidin-3'-yl]ethane-1,2-diamine
wild type eNOS
0.0952
N1-[trans-4'-[(6''-amino-4''-methylpyridin-2''-yl)methyl]pyrrolidin-3'-yl]-N2-(3'-chlorobenzyl)ethane-1,2-diamine
wild type eNOS
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
assay at
7.6
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
role of oxidative stress in the dysfunction of the placental endothelial nitric oxide synthase in preeclampsia (PE), multifactorial pregnancy disease, characterized by new-onset gestational hypertension with (or without) proteinuria or end-organ failure, exclusively observed in humans. PE pathophysiology can result from abnormal placentation due to a defective trophoblastic invasion and an impaired remodeling of uterine spiral arteries, leading to a poor adaptation of utero-placental circulation. This would be associated with hypoxia/ reoxygenation phenomena, oxygen gradient fluctuations, altered antioxidant capacity, oxidative stress, and reduced nitric oxide (NO) bioavailability. This results in part from the reaction of NO with the radical anion superoxide, which produces peroxynitrite ONOO-, a powerful pro-oxidant and inflammatory agent. Another mechanism is the progressive inhibition of the placental endothelial nitric oxide synthase (eNOS) by oxidative stress, which results in eNOS uncoupling via several events such as a depletion of the eNOS substrate L-arginine due to increased arginase activity, an oxidation of the eNOS cofactor tetrahydrobiopterin (BH4), or eNOS posttranslational modifications (for instance by S-glutathionylation). The uncoupling of eNOS triggers a switch of its activity from a NO-producing enzyme to a NADPH oxidase-like system generating superoxide, thereby potentiating ROS production and oxidative stress. Moreover, in PE placentas, eNOS can be posttranslationally modified by lipid peroxidation-derived aldehydes such as 4-oxononenal (ONE) a highly bioreactive agent, able to inhibit eNOS activity and NO production. Analysis of the dysfunction of placental eNOS evoked by oxidative stress and lipid peroxidation products, and the potential consequences on PE pathogenesis, detailed overview. Oxidative stress is thought to play a pivotal role in the decreased NO bioavailability in PE pathophysiology, via several mechanisms including an inhibition of eNOS (eNOS uncoupling) and subsequent defect of NO biosynthesis, or through the formation of peroxynitrite, via the reaction of NO with the radical anion superoxide. eNOS inhibition is associated with a decrease in endothelial-dependent relaxation in vitro and in vivo. Therapeutic perspectives targeting oxidative stress and NO/eNOS dysfunction
physiological function
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
NOS3_HUMAN
1203
0
133275
Swiss-Prot
other Location (Reliability: 2)
PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
in PE placentas, eNOS can be posttranslationally modified by lipid peroxidation-derived aldehydes such as malondialdehyde or as 4-oxononenal (ONE) a highly bioreactive agent, able to inhibit eNOS activity and NO production. They covalently bind to the nucleophilic sulfhydryl and primary amine groups of proteins, forming Schiff bases, Michael adducts and protein crosslinks. The modification of proteins by lipid peroxidation products (LPPs) depends on their nature, expression and conformation, oxidative stress intensity and duration, cell type, local LPP concentration, and generates various biological responses from the expression of protective and adaptive factors to protein dysfunction, inflammation, senescence and apoptosis. The presence of LPPs in PE placentas, could be indicative of their premature senescence, in agreement with the hypothesis that accelerated placental aging is involved in PE pathophysiology via oxidative stress
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G2A
the mutant is defective in activity and cellular localization
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni Sepharose column chromatography, 2',5'-ADP Sepharose column chromatography, and Superdex 200 gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of eNOS mRNA and protein does not change in mild to moderate chronic obstructive pulmonary disease, but is reduced in the more severe stages of chronic obstructive pulmonary disease
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ghosh, D.K.; Holliday, M.A.; Thomas, C.; Weinberg, J.B.; Smith, S.M.; Salerno, J.C.
Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs
J. Biol. Chem.
281
14173-14183
2006
Mus musculus, Homo sapiens (P29474), Rattus norvegicus (P29476)
Manually annotated by BRENDA team
Brindicci, C.; Kharitonov, S.A.; Ito, M.; Elliott, M.W.; Hogg, J.C.; Barnes, P.J.; Ito, K.
Nitric oxide synthase isoenzyme expression and activity in peripheral lungs of COPD patients
Am. J. Respir. Crit. Care Med.
181
21-30
2009
Homo sapiens (P29474), Homo sapiens (P29475), Homo sapiens (P35228), Homo sapiens
Manually annotated by BRENDA team
Mikula, I.; Durocher, S.; Martasek, P.; Mutus, B.; Slama-Schwok, A.
Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia
Biochem. J.
418
673-682
2009
Homo sapiens, Homo sapiens (P29474), Homo sapiens (P29475)
Manually annotated by BRENDA team
Lenasi, H.; Strucl, M.
The effect of nitric oxide synthase and cyclooxygenase inhibition on cutaneous microvascular reactivity
Eur. J. Appl. Physiol.
103
719-726
2008
Homo sapiens (P29474), Homo sapiens
Manually annotated by BRENDA team
Igarashi, J.; Li, H.; Jamal, J.; Ji, H.; Fang, J.; Lawton, G.R.; Silverman, R.B.; Poulos, T.L.
Crystal structures of constitutive nitric oxide synthases in complex with de novo designed inhibitors
J. Med. Chem.
52
2060-2066
2009
Homo sapiens (P29474), Homo sapiens (P29475)
Manually annotated by BRENDA team
Ibiza, S.; Perez-Rodriguez, A.; Ortega, A.; Martinez-Ruiz, A.; Barreiro, O.; Garcia-Dominguez, C.A.; Victor, V.M.; Esplugues, J.V.; Rojas, J.M.; Sanchez-Madrid, F.; Serrador, J.M.
Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells
Proc. Natl. Acad. Sci. USA
105
10507-10512
2008
Homo sapiens (P29474)
Manually annotated by BRENDA team
Chennoufi, R.; Cabrie, A.; Nguyen, N.H.; Bogliotti, N.; Simon, F.; Cinquin, B.; Tauc, P.; Boucher, J.L.; Slama-Schwok, A.; Xie, J.; Deprez, E.
Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase
Biochim. Biophys. Acta
1863
1127-1137
2019
Homo sapiens (P29474)
Manually annotated by BRENDA team
Guerby, P.; Tasta, O.; Swiader, A.; Pont, F.; Bujold, E.; Parant, O.; Vayssiere, C.; Salvayre, R.; Negre-Salvayre, A.
Role of oxidative stress in the dysfunction of the placental endothelial nitric oxide synthase in preeclampsia
Redox Biol.
40
101861
2021
Homo sapiens (P29474), Homo sapiens
Manually annotated by BRENDA team