Information on EC 1.13.11.61 - linolenate 9R-lipoxygenase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.13.11.61
-
RECOMMENDED NAME
GeneOntology No.
linolenate 9R-lipoxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
alpha-linolenate + O2 = (9R,10E,12Z,15Z)-9-hydroperoxyoctadeca-10,12,15-trienoate
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
alpha-linolenate:oxygen (9R)-oxidoreductase
In cyanobacteria the enzyme is involved in oxylipin biosynthesis. The enzyme also converts linoleate to (9R,10E,12Z)-9-hydroperoxyoctadeca-10,12-dienoate.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
structure-activity analysis, overview. Crucial residues in the direct environment of the narrow active site that form a clamp-like structure include Tyr360, Gly401 Ile617, Ile379, Leu405, and Leu621. Molecular dynamics simulations support a major role of steric shielding of active site clamp. Structure-activity analysis, modeling, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
alpha-linolenate + O2
(9R,10E,12Z,15Z)-9-hydroperoxy-10,12,15-octadecatrienoate
show the reaction diagram
alpha-linolenic acid + O2
(9R,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid
show the reaction diagram
alpha-linolenic acid + O2
(9R,10E,12Z,15Z)-9-hydroxy-octadeca-10,12,15-trienoic acid
show the reaction diagram
gamma-linolenic acid + O2
(6Z,9R,10E,12Z)-9-hydroxy-octadeca-6,10,12-trienoic acid
show the reaction diagram
linoleate + O2
(9R,10E,12Z)-9-hydroperoxy-10,12-octadecadienoate
show the reaction diagram
linoleate + O2
(9Z,12Z)-(9R)-9-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
linoleic acid + O2
(9R,10E,12Z)-9-hydroxy-octadeca-10,12-dienoic acid
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
alpha-linolenate + O2
(9R,10E,12Z,15Z)-9-hydroperoxy-10,12,15-octadecatrienoate
show the reaction diagram
-
i.e. (9Z,12Z,15Z)-octadeca-9,12,15-trienoate
-
-
?
alpha-linolenic acid + O2
(9R,10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid
show the reaction diagram
gamma-linolenic acid + O2
(6Z,9R,10E,12Z)-9-hydroxy-octadeca-6,10,12-trienoic acid
show the reaction diagram
linoleate + O2
(9R,10E,12Z)-9-hydroperoxy-10,12-octadecadienoate
show the reaction diagram
-
the enzyme is involved in oxylipin biosyntesis
-
-
?
linoleate + O2
(9Z,12Z)-(9R)-9-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
-
-
-
-
?
linoleic acid + O2
(9R,10E,12Z)-9-hydroxy-octadeca-10,12-dienoic acid
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
iron cofactor
-
coordination of the iron cofactor, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe3+
-
non-heme iron, required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
SDS
-
-
SPAN 20
-
slight inhibition
-
Span 40
-
slight inhibition
-
Span 60
-
slight inhibition
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SPAN 80
-
slight inhibition
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Triton X-100
-
-
additional information
-
no effect by Brij58; no effects by Brij58
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetone
-
activates best at 5% v/v
Tween 20
-
-
Tween 40
-
-
Tween 80
-
activates best at 0.2% w/v
additional information
-
no effect by Brij58
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.026
alpha-linolenate
-
pH 7.5, 22C, wild-type enzyme
0.054
alpha-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
0.054
gamma-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
0.0044 - 0.0089
linoleate
0.404
linoleic acid
-
recombinant enzyme, pH 8.5, 15C
additional information
additional information
-
Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.13
alpha-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
0.097
gamma-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
5.12
linoleic acid
-
recombinant enzyme, pH 8.5, 15C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.4
alpha-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
1.82
gamma-linolenic acid
-
recombinant enzyme, pH 8.5, 15C
12.67
linoleic acid
-
recombinant enzyme, pH 8.5, 15C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.4
-
recombinant enzyme, pH 8.5, 15C, substrate gamma-linolenic acid
6.3
-
recombinant enzyme, pH 8.5, 15C, substrate alpha-linolenic acid
18.4
-
recombinant enzyme, pH 8.5, 15C, substrate linolenic acid
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10
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broad pH-optimum
7.3
-
assay at
8.5
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 10.5
-
50% of maximal activity at pH 4.5-6.5, maximal activity at pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70900
-
x * 70900, calculated from sequence
156000
-
recombinant His-tagged enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 70900, calculated from sequence
homotrimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type enzyme CspLOX2, sitting drop vapor diffusion method, from well solution of 10% PEG 4000, 0.1 M MES/imidazole pH 6.5, 20% glycerol and 0.02% alcohols (1,6-hexanediol, 1-butanol, 1,2-propanediol, 2-propanol, 1,4-butanediol, and 1,3-propanediol), 10 days, purified recombinant CspLOX2 mutant enzymes by hanging drop vapour diffusion method, from well solution of 12.5-15 PEG 4000, 8-12% glycerol, 0.1 M MES/imidazole pH 6.1, and 0-600 mM NaCl, X-ray diffraction structure determination and analysis at 1.8 A resolution. Crystals of a CspLOX2 substrate complex are not obtained
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant carboxy-terminal domain is purified after expression in Escherichia coli
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recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography
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recombinant His-tagged enzyme from Escherichia coli strain BL21 Star by nickel affinity chromatography, gel filtration, and ultrafiltration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of separate domains of NspLOX and the entire protein in Escherichia coli
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gene 9R-lox, recombinant expression of His-tagged enzyme in Escherichia coli
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recombinant carboxy-terminal domain is purified after expression in Escherichia coli
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recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 Star
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G401A
-
site-directed mutagenesis, the mutaant shows increased formation of 11-hydroperoxyoctadeca-9,12-dienoate compared to the wild-type enzyme
I617L
-
site-directed mutagenesis, the mutaant shows increased formation of 11-hydroperoxyoctadeca-9,12-dienoate compared to the wild-type enzyme
A162G
-
the recombinant carboxy-terminal domain of the wild-type enzyme and of mutant enzyme A162G produce primarily (9R,10EZ,12Z)-9-hydroperoxy-10,12-octadecadienoate from linoleate. The stereochemistry of the hydroperoxide is almost exclusively R (93-94%)
A162I
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the recombinant carboxy-terminal domain of the mutant enzyme A162I produces almost exlusively (13S)-hydroperoxy octadecadienoic acid (90%)
A162V
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the recombinant carboxy-terminal domain of the mutant enzyme A162V converts linoleate primarily to (13S)-hydroperoxy octadecadienoic acid (64%) and to a lesser extent to (9R,10EZ,12Z)-9-hydroperoxy-10,12-octadecadienoate (36%)
additional information
-
Decreasing the channel size of a 9R-lipoxygenase (CspLOX1) can in turn induce formation of the bis-allylic 11R-hydroperoxide