Information on EC 1.13.11.60 - linoleate 8R-lipoxygenase

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The expected taxonomic range for this enzyme is: leotiomyceta

EC NUMBER
COMMENTARY hide
1.13.11.60
-
RECOMMENDED NAME
GeneOntology No.
linoleate 8R-lipoxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
linoleate + O2 = (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
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-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Linoleic acid metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
linoleate:oxygen (8R)-oxidoreductase
The enzyme contains heme [1,4]. The bifunctional enzyme from Aspergillus nidulans uses different heme domains to catalyse two separate reactions. Linoleic acid is oxidized within the N-terminal heme peroxidase domain to (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, which is subsequently isomerized by the C-terminal P-450 heme thiolate domain to (5S,8R,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate (cf. EC 5.4.4.5, 9,12-octadecadienoate 8-hydroperoxide 8R-isomerase) [1]. The bifunctional enzyme from Gaeumannomyces graminis also catalyses the oxidation of linoleic acid to (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, but its second domain isomerizes it to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate (cf. EC 5.4.4.6, 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase) [4].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase and hydroperoxide isomerase activities, respectively
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(11Z)-icos-11-enoic acid + O2
10-hydroperoxyeicosenoic acid
show the reaction diagram
-
-
-
?
(11Z)-icosa-11-enoate + O2
?
show the reaction diagram
-
-
-
?
(11Z,14Z)-icosa-11,14-dienoic acid + O2
(11Z,14Z)-10-hydroperoxyeicosa-11,14-dienoic acid
show the reaction diagram
-
-
-
?
(11Z,14Z)-icosa-11,14-dienoic acid + O2
?
show the reaction diagram
-
-
-
?
(11Z,14Z,17Z)-icosa-11,14,17-trienoic acid + O2
(11Z,14Z,17Z)-10-hydroperoxyeicosa-11,14,17-trienoic acid
show the reaction diagram
-
-
-
?
alpha-linolenate + O2
?
show the reaction diagram
-
-
-
?
elaidate + O2
?
show the reaction diagram
-
-
-
?
linoleate + O2
(8R,10E,12Z)-8-hydroperoxy-10,12-octadecadienoate
show the reaction diagram
linoleate + O2
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
show the reaction diagram
linoleate + O2
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
oleate + O2
(9Z)-8-hydroperoxyoctadec-9-enoate
show the reaction diagram
-
-
-
?
palmitoleate + O2
(9Z)-8-hydroperoxyhexadec-9-enoate
show the reaction diagram
-
-
-
?
palmitoleate + O2
?
show the reaction diagram
-
-
-
?
additional information
?
-
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
linoleate + O2
(8R,10E,12Z)-8-hydroperoxy-10,12-octadecadienoate
show the reaction diagram
-
-
-
-
?
linoleate + O2
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
show the reaction diagram
linoleate + O2
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
C1KH66
all-cis-9,12-octadecadienoic acid
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome P450
PpoA contains a high spin ferriheme. PpoA uses different heme domains to catalyze two separate reactions. Within the N-terminal heme peroxidase domain, linoleic acid is oxidized to (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate is isomerized within the C-terminal P450 heme thiolate domain to (5S,8R,9Z,12Z)-5,8-dihydroxy-9,12-octadecadienoate
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heme
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ferric hemeprotein. The proximal heme ligand of linoleate diol synthase is tentatively identified as His379 and the important tyrosine for catalysis as residue376 (apparent consensus EFNXXXYXWH). The distal heme ligand is tentatively identified as His203 (apparent consensus THXXFXT)
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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2.5 mM, 50% inhibition
2-mercaptoethanol
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-
4-aminobenzamidine
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1 mM, over 70% inhibition
acetonitrile
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10%, inactivation
diclofenac
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0.2 mM, over 80% inhibition
diethyldicarbonate
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-
dithiothreitol
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-
eicosatetraynoic acid
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-
Emulgen 911
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0.5%, inactivation
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ICI 230,487
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0.03 mM, 58% inhibition
Isopropanol
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10%, inactivation
linoleate-hydroxamic acid
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-
linoleic acid
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0.1 mM, partial inactivation, may indicate product inhibition
N-tosyl-L-phenylalanyl chloromethyl ketone
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1 mM, over 70% inhibition
N-[(E)-3-(3-phenoxyphenyl)prop-2-enyl]acetohydroxamic acid
nordihydroguaiaretic acid
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paracetamol
phenylmethanesulfonyl fluoride
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0.2 mM, inhibits the 8R-dioxygenase enzyme by over 85%
polyoxyethylene-10 lauryl ether
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0.5%, inactivation
Sodium deoxycholate
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0.5%, inactivation
Tetranitromethane
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0.4 mM, 50% inhibition
Triton X-100
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0.5%, inactivation
Tween 20
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0.5%, inactivation
Tween 80
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0.5%, inactivation
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CHAPS
-
0.5 mM, augments enzyme activity
Tween 20
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0.04%, augments enzyme activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0226
(11Z)-icos-11-enoic acid
pH 7.2, 24C
0.008 - 0.0183
linoleate
0.03
O2
-
pH 7.4 25C
0.00671
oleate
pH 7.2, 24C
0.005
palmitoleate
pH 7.2, 24C
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.9
(11Z)-icos-11-enoic acid
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
6.3 - 15
linoleate
4.95
oleate
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
3.5
palmitoleate
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
261
(11Z)-icos-11-enoic acid
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
39642
344
linoleate
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
808
738
oleate
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
488
700
palmitoleate
Aspergillus nidulans
Q6RET3
pH 7.2, 24C
5594
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1
dithiothreitol
Gaeumannomyces graminis
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pH and temperature not specified in the publication
0.15
eicosatetraynoic acid
Gaeumannomyces graminis
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pH and temperature not specified in the publication
0.02
linoleate-hydroxamic acid
Gaeumannomyces graminis
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pH and temperature not specified in the publication
0.0002 - 0.002
N-[(E)-3-(3-phenoxyphenyl)prop-2-enyl]acetohydroxamic acid
0.09
nordihydroguaiaretic acid
Gaeumannomyces graminis
-
pH and temperature not specified in the publication
0.003 - 0.01
zileuton
Gaeumannomyces graminis
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pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.8
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pH 7.4, 0C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.2
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chromatofocusing
6.1 - 6.3
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
440000
gel filtration
520000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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7,8-LDS contains five tentative N-linked glycosylation sites, one of which has high probability of glycosylation. This N-glycosylation site, Asn216, is located near the distal heme-binding region along with tentative sites for O-linked glycosylation. Asn216Gln shows no detectable enzyme activity, although expression of Asn216Gln is confirmed by Western blot analysis. alpha-Mannosidase treatment does not change the 8R-dioxygenase activity but abolishes the hydroperoxide isomerase activity. Proper N- and O-linked glycosylation could be important for the hydroperoxide isomerase. Expression of 7,8-LDS in insect cells yields recombinant 7,8-LDS with the native hydroperoxide isomerase activity
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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stable
285247
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
8R-dioxygenase is unstable during purification. It can be stabilized by glutathione, glutathione peroxidase and ethylenediaminetetraacetic acid
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repeated freezing and thawing, particularly of dilute enzyme solutions, inactivates the enzyme
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the enzyme is quite unstable at the early steps of purification, but active fractions from anion-exchange chromatography can be kept for 1 week on ice with only a small loss of enzyme activity
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
urea
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the enzyme can be incubated in 4 M urea for 1 h, followed by desalting, with only partial loss of enzyme activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Spodoptera frugiperda (Sf21) insect cells with native 8R-dioxygenase and hydroperoxide isomerase activities
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expression in Escherichia coli
expression in Escherichia coli and in insect cells
expression in Pichia pastoris changes the position and stereospecificity of a hydroperoxide isomerase possibly due to N- or O-linked mannosides in the vicinity of the heme group, whereas the 8(R)-dioxygenase activity is identical with native 7,8-linoleate diol synthase
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expression in SF21 cells
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expression of wild-type and mutant enzymes in Escherichia coli strain BL21
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C1006S
the hydroperoxide isomerase activity of 5,8-LDS mutant is abolished
C1006S/V328F
the mutant forms after reduction of hydroperoxides to alcohols, (8R)-hydroperoxy-(9Z,12Z)-octadecadienoic acid and (10R)-hydroxy-(8E,12Z)-octadecadienoic acid in the same relative amounts
C1006S/V328L
the mutant forms after reduction of hydroperoxides to alcohols, (8R)-hydroperoxy-(9Z,12Z)-octadecadienoic acid and (10R)-hydroxy-(8E,12Z)-octadecadienoic acid in the same relative amounts
C1006S/V328S
the mutant forms after reduction of hydroperoxides to alcohols, (8R)-hydroperoxy-(9Z,12Z)-octadecadienoic acid and (10R)-hydroxy-(8E,12Z)-octadecadienoic acid in the same relative amounts
N887L
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site-directed mutagenesis, the mutant retains (5S,8R)-5,8-dihydroxylinoleate as the main metabolite with an increased formation of 6,8- and 8,11-dihydroxylinoleate
N887Q
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site-directed mutagenesis, the mutant retains (5S,8R)-5,8-dihydroxylinoleate as the main metabolite with an increased formation of 6,8- and 8,11-dihydroxylinoleate
Q890E
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site-directed mutagenesis, the mutant retains (5S,8R)-5,8-dihydroxylinoleate as the main product, but shifts oxygenation from C-5 to C-7 and C-11 and to formation of epoxyalcohols by homolytic scission of (8R)-hydroperoxylinoleic acid
Q890L
-
site-directed mutagenesis, the mutant shows almost abolished 5,8-linoleate diol synthase activity
Y374F
mutation in the conserved sequence YRWH results in loss of linoleate 8R-lipoxygenase activity, whereas the hydroperoxide isomerase activity is retained
H1004A
mutant enzyme with abolished 8-hydroperoxide isomerase activity, whereas dioxygenase activity is still present
Y374
the mutant enzyme shows no detectable activity when incubated with (9Z,12Z)-octadeca-9,12-dienoate as a substrate. When incubated with the intermediate product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate it is able to catalyze isomerization to (5S,8R,9Z,12Z)-5,8-dihydroperoxy-9,12-octadecadienoate
E384A
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mutant forms only traces of (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
H203Q
-
weak activity
I375A
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mutant has virtually no dioxygenase activity
K540L
-
inactive mutant enzyme
K540Q
-
inactive mutant enzyme
K540R
-
inactive mutant enzyme
L334V
-
inactive mutant enzyme
N216Q
-
inactive mutant enzyme, no 8(R)-dioxygenase activity
N938D
-
site-directed mutagenesis, the mutation of the 7,8-linoleate diol synthase reduces the hydroperoxide isomerase activity of the enzyme
N938L
-
site-directed mutagenesis, the mutation of the 7,8-linoleate diol synthase abolishes the hydroperoxide isomerase activity of the enzyme
N938Q
-
site-directed mutagenesis, the mutation of the 7,8-linoleate diol synthase reduces the hydroperoxide isomerase activity of the enzyme
V330L
-
the wild-type enzyme forms 98% (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate and 2% (8E,10R,12Z)-10-hydroperoxy-9,12-octadecadienoate. The V330L mutation augments the formation of (8E,10R,12Z)-10-hydroperoxy-8,12-octadecadienoate 3fold
Y329F
-
weak activity
Y329L
-
inactive mutant enzyme
Y376F
-
inactive mutant enzyme
Y378F
-
inactive mutant enzyme
Y378S
-
inactive mutant enzyme
Y531F
-
inactive mutant enzyme
additional information