Information on EC 1.13.11.40 - arachidonate 8-lipoxygenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.13.11.40
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RECOMMENDED NAME
GeneOntology No.
arachidonate 8-lipoxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
arachidonate + O2 = (5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
from the coral Pseudoplexaura porosa
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dioxygenation
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oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arachidonic acid metabolism
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arachidonic acid metabolism
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SYSTEMATIC NAME
IUBMB Comments
arachidonate:oxygen 8-oxidoreductase
From the coral Pseudoplexaura porosa.
CAS REGISTRY NUMBER
COMMENTARY hide
100900-72-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
marine mollusc
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Manually annotated by BRENDA team
starfish
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Manually annotated by BRENDA team
clam, synonym Concha fina
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Manually annotated by BRENDA team
gorgonian coral
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Manually annotated by BRENDA team
sea urchin
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(15S)-hydroperoxyeicosatetraenoic acid + O2
?
show the reaction diagram
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-
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?
(15S)-hydroxyeicosatetraenoic acid + O2
?
show the reaction diagram
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-
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?
(5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid + O2
?
show the reaction diagram
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8-LOX tolerates a carboxylic group in the substrate-binding pocket
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?
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid + O2
(8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicosatetraenoic acid
show the reaction diagram
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?
(8S)-hydroxyeicosatetraenoic acid + O2
?
show the reaction diagram
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?
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
show the reaction diagram
5-hydroperoxy fatty acid + O2
leukotriene A4
show the reaction diagram
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leukotriene A synthase activity, reaction rate is approximately 7% of arachidonate 8-lipoxygenation
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?
5-hydroperoxyeicosatetraenoic acid + ?
leukotriene A4
show the reaction diagram
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unstable product
?
alpha-linolenic acid + O2
?
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonic acid + O2
(5Z,8E,11Z,14Z)-8-hydroperoxyicosa-5,8,11,14-tetraenoic acid + (5Z,8E,11Z,14Z)-8-hydroxyicosa-5,8,11,14-tetraenoic acid
show the reaction diagram
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arachidonic acid, enzyme activity only detectable after in vivo treatment with the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate)
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?
arachidonic acid + O2
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
show the reaction diagram
docosahexaenoic acid + O2
?
show the reaction diagram
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?
eicosapentaenoic acid + O2
?
show the reaction diagram
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?
gamma-linoleic acid + O2
?
show the reaction diagram
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?
linoleic acid + O2
?
show the reaction diagram
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poor substrate
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?
additional information
?
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the inducible expression of 8-lipoxygenase inhibits cell growth, 15-LOX-2 and 8-LOX, although displaying different positional specificity, use common signaling pathways to induce growth inhibition in premalignant epithelial cells, overview
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2 arachidonic acid + 2 O2
8-hydroxy,9-oxo-eicosa-5Z,11Z,14Z-trienoic acid + 9-oxo-[8,12-cis]-prosta-5Z,10,14Z-trienoic acid + H2O
show the reaction diagram
arachidonate + O2
(5Z,9E,11Z,14Z)-(8R)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyeicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
arachidonate + O2
(5Z,9E,11Z,14Z)-(8S)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
show the reaction diagram
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?
additional information
?
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the inducible expression of 8-lipoxygenase inhibits cell growth, 15-LOX-2 and 8-LOX, although displaying different positional specificity, use common signaling pathways to induce growth inhibition in premalignant epithelial cells, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
AA861
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baicalein
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beta-mercaptoethanol
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Eicosa-5,8,11,14-tetraynoic acid
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glutathione
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50% inhibition
nordihydroguaiaretic acid
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additional information
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physico-chemical state of the substrate and the complex equilibrium between fatty acid monomers, acid soaps and micelles may impact the reaction specificity of LOX-isoforms
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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lecithin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.039
(15S)-hydroperoxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.015
(15S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.0021
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.0057
(8S)-hydroxyeicosatetraenoic acid
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.008 - 0.135
arachidonate
0.0012 - 0.008
arachidonic acid
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
(15S)-hydroperoxyeicosatetraenoic acid
Mus musculus
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
1
(15S)-hydroxyeicosatetraenoic acid
Mus musculus
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
7
(8R)-8-hydroperoxy-5,9,11,14-eicosatetraenoic acid
Mus musculus
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
3.5
(8S)-hydroxyeicosatetraenoic acid
Mus musculus
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in 50 mM sodium phosphate buffer, pH 7.4, at 25°C
0.3 - 206
arachidonate
3.2 - 66.7
arachidonic acid
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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specific activity of 8-LOX with (5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid as substrate strongly increases going from pH 6 to 7.2 but then drops down at more alkaline pH
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8
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8
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8-LOX
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9.5
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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nervous tissue
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
76000
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mature protein predicted from the cDNA, difference from the size estimated by SDS-PAGE implies a post-translational modification of the enzyme
100000
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SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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different values obtained from cDNA and SDS-PAGE imply a post-transitional modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
deletion mutant of 8R-LOX crystallized by sitting drop vapor diffusion, to 1.85 A resolution, belongs to space group P21 with four molecules in the asymmetric unit. U-shaped channel in 8R-LOX
hanging drop vapour diffusion in 6.25% polyethylene glycol 8000, 100 mM imidazole acetate (pH 8.0), 100 mM CaCl2, and 5% sucrose at 22°C
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
leukotriene A synthase activity of the enzyme leveled off within 10 min, indicating suicide activation
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unstable in solution, activity is completely lost after standing on ice overnight
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, crude and purified enzyme preparations can be stored without appreciable loss of activity for at least 6 months
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-80°C, 10% glycerol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA-agarose column chromatography
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nickel-nitrilotriacetic acid-agarose chromatography, DE52 column chromatography, and Mono Q column chromatography
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on Ni column
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recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli strain BL21 (DE3)
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expression and establishment of a doxycycline-inducible Tet-On gene expression system in the premalignant mouse keratinocyte cell line 308
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expression in HeLa cells
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full-length cDNA cloned by PCR
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ligated into the pQE-9 plasmid and expressed as N-terminal His-tag fusion protein in Escherichia coli (XL-1 Blue)
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skin 8-lipoxygenase expressed in COS-7 cells by transient transfection of its cDNA, amplified by PCR, in pEF-BOS, also expressed in Escherichia coli system using pQE-32
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H604F
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8-LOX mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation product. Specific activity of 8-LOX mutant with (5Z,8Z,11Z,14Z)-nonadeca-5,8,11,14-tetraene-1,19-dioic acid as substrate continously declines when pH increases
Y603F
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8-LOX mutant, is relative insensitive towards pH alterations in the near physiological range (pH 6-8), at strong alkaline conditions (pH more than 9) significant shares of 15S-H(p)ETE are formed
Y603F/H604F
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8-LOX double mutant, induces strong pH-dependent alterations in the positional specificity but the pH-optimum remains the same. At acidic pH 8S-H(p)ETE is the exclusive arachidonic acid oxygenation product but with more alkaline pH increasing shares of 15S-H(p)ETE, above pH 9 15S-H(p)ETE is the major oxygenation product
A417G
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converts arachidonic acid mainly to 12-hydroxyeicosatetraenoic acid, mutant retains 38% of catalytic efficiency
A417S
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same oxygenase specificity and similar catalytic activity to wild-type 8S-LOX
D39A
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118% activity compared to the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
D39A/E47A
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106% activity compared to the wild type enzyme, a double mutant with calcium-binding residues from two of the three sites mutated exhibits no fluorescence resonance energy transfer signal
E47A
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65% of the activity of the wild type enzyme, mutant with diminished fluorescence resonance energy transfer properties, consistent with a role for calcium in membrane binding
I433A
absence of the Ile side chain destabilizes the roof of the U-shaped channel, measurable activity only in the presence of CaCl2 and the detergent emolphogen
I433W
has no measurable activity, presumably because the Trp side chain effectively blocks the arachidonic acid binding site
L432A
less than 5% of the activity of the wild-type
L432F
less than 5% of the activity of the wild-type
L432I
less than 5% of the activity of the wild-type
L432V
less than 20% of the activity of the wild-type
W41A
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140% activity compared to the wild type enzyme, exhibits only less than 2% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
W77A
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44% of the activity of the wild type enzyme, exhibits only 4% of the increase in fluorescence at 517 nm upon the addition of CaCl2 of the wild-type signal
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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therapeutic effect of inhibitors of arachidonic acid metabolism on inflammatory skin diseases and epidermal tumors and on tumor development