Information on EC 1.13.11.45 - linoleate 11-lipoxygenase

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

EC NUMBER
COMMENTARY hide
1.13.11.45
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RECOMMENDED NAME
GeneOntology No.
linoleate 11-lipoxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
linoleate + O2 = (9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Linoleic acid metabolism
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SYSTEMATIC NAME
IUBMB Comments
linoleate:oxygen 11S-oxidoreductase
The product (9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate, is converted, more slowly, into (9Z,11E)-(13R)-13-hydroperoxyoctadeca-9,11-dienoate. The enzyme from the fungus Gaeumannomyces graminis requires Mn2+. It also acts on alpha-linolenate, whereas gamma-linolenate is a poor substrate. Oleate and arachidonate are not substrates.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-60-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
navy bean
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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
(10Z,13Z,16Z)-nonadeca-10,13,16-trienoic acid + O2
?
show the reaction diagram
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oxidation at the n-6 carbon and at the bis-allylic position n-8
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-
?
(11Z,14Z)-eicosa-11,14-dienoic acid + O2
?
show the reaction diagram
-
LC-MS analysis shows that Fo-MnLOX oxidizes (11Z,14Z)-eicosa-11,14-dienoic acid efficiently at both C-15 and C-11
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-
?
(11Z,14Z)-eicosadienoic acid + O2
15-hydroxyperoxy-(9Z,11E)-eicosadienoic acid
show the reaction diagram
-
-
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?
(11Z,14Z,17Z)-eicosa-11,14,17-trienoic acid + O2
15-hydroxyperoxy-11Z,13E,17Z-eicosatrienoic acid
show the reaction diagram
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via 11-hydroperoxyoctadecadienoic acid, 13-hydroperoxyoctadecadienoic acid and 15-hydroperoxyoctadecadienoic acid
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-
?
(11Z,14Z,17Z)-eicosa-11,14,17-trienoic acid + O2
?
show the reaction diagram
(13R)-hydroperoxylinolenic acid + O2
13-ketolinolenic acid + epoxyalcohols
show the reaction diagram
only mutant enzyme G316A
erythro- and threo-11-hydroxy-(12R,13R)-epoxy-(9Z,15Z)-octadecadienoic acids and one of the corresponding cis-epoxides as major products
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?
(5E,8E,11E,14E)-icosa-5,8,11,14-tetraenoic acid + O2
(5Z,8Z,11S,12E,14Z)-11-hydroperoxyicosa-5,8,12,14-tetraenoic acid + (5Z,8Z,11Z,13E,15R)-15-hydroperoxyicosa-5,8,11,13-tetraenoic acid
show the reaction diagram
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oxidation at the n-6 carbon and at the bis-allylic position n-8
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?
(5Z,8Z,11Z,14Z,17Z)-docosa-5,8,11,14,17-pentaenoate + O2
?
show the reaction diagram
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oxidation at the n-6 carbon and at the bis-allylic position n-8
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-
?
(7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoate + O2
?
show the reaction diagram
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oxidation at the n-6 carbon and at the bis-allylic position n-8
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?
(9E,12Z)-octadeca-9,12-dienoic acid + O2
?
show the reaction diagram
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sLOX-1 oxidizes (9E,12Z)18:2 to 9-hydroperoxyoctadecadienoic acid in an R/S ratio of 60/40 and to 13-hydroperoxyoctadecadienoic acid
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?
(9Z,11S,12Z)-11-hydroperoxyoctadeca-9,12-dienoate + O2
(9Z,11E, 13R)-13-hydroperoxyoctadeca-9,11-dienoate
show the reaction diagram
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?
(9Z,11S,12Z)-11-hydroperoxyoctadeca-9,12-dienoate + O2
(9Z,11E,13R)-13-hydroperoxyoctadeca-9,11-dienoate
show the reaction diagram
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?
(9Z,12E)-octadeca-9,12-dienoic acid + O2
?
show the reaction diagram
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sLOX-1 oxidizes (9Z,12E)18:2 slowly to the 9-hydroperoxy metabolite with 10E,12E configuration as the main product (65%) and to 13-hydroperoxyoctadecadienoic acid
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?
(9Z,12Z)-octadeca-9,12-dienoic acid + O2
(9S)-hydroperoxy-octadeca-10,12-dienoate
show the reaction diagram
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alpha-linoleate is converted via two intermediates, (11S)-hydroperoxy-(9Z,12Z)-octadecenoate and (13R)-hydroperoxy-(9Z,11E)-octadecadienoate, which are isomerized to the end product, probably after oxidation to peroxyl radicals, beta-fragmentation, and oxygen insertion at C-9
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?
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid + O2
(10E,12E,14E)-9,16-dihydroxy-octadeca-10,12,14-trienoate
show the reaction diagram
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gamma-linoleate is oxidized at C-9, C-11, and C-13
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?
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid + O2
(13R)-hydroperoxyoctadecadienoic acid
show the reaction diagram
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via bis-allylic 11S-hydroperoxy fatty acid
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?
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid + O2
?
show the reaction diagram
1,2-dilinoleoyl-phosphatidylcholine + O2
?
show the reaction diagram
a poor substrate
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?
1,2-dilinoleoyl-sn-glycero-3-phosphocholine + O2
?
show the reaction diagram
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main products with 14% and 10%, no production of 11-hydroxyoctadecanoic acid
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?
1-linoleoyl-2-hydroxy-phosphatidylcholine + O2
?
show the reaction diagram
an excellent substrate
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?
1-linoleoyl-2-lyso-phosphatidylcholine + O2
?
show the reaction diagram
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?
1-linoleyl-lyso-glycerophosphatidylcholine + O2
11-hydroxyoctadecanoic acid + 13-hydroxyoctadecanoic acid
show the reaction diagram
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via formation of the 13-hydroxyperoxy metabolite
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?
11-(11R)-hydroperoxy-(9Z,12Z)-octadecadienoic acid
9-(9R)-hydroperoxy-octadecadienoic acid
show the reaction diagram
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slow
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?
11-(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid
13-(13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid
show the reaction diagram
11-(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid methyl ester
13-(13R)-hydroperoxy-(9Z,11E)-octadecadienoic acid methyl ester
show the reaction diagram
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?
11-hydroperoxyoctadecadienoic acid + O2
11,18-dihydroxy-12E,14Z,16E-eicosatrienoic acid
show the reaction diagram
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two diastereoisomers
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?
alpha-linolenic acid + O2
(13R)-hydroperoxy octadecadienoic acid + 9-hydroperoxyoctadecadienoic acid
show the reaction diagram
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?
alpha-linolenic acid + O2
(9Z,11E,15Z)-(13)-hydroperoxyoctadecatrienoic acid
show the reaction diagram
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?
alpha-linolenic acid + O2
?
show the reaction diagram
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?
gamma-linolenic acid + O2
(6Z,9Z,11E)-(13)-hydroperoxyoctadecatrienoic acid + (6Z,9Z,12Z)-(11)-hydroperoxyoctadecatrienoic acid
show the reaction diagram
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?
linoleate + O2
(9Z,11S,12Z)-11-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
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?
linoleate + O2
(9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
linoleic acid + O2
(9Z,11E)-(13R)-hydroperoxyoctadecadienoic acid
show the reaction diagram
linoleic acid + O2
(9Z,11E)-(13R)-hydroperoxyoctadecadienoic acid + (9Z,12Z)-(11S)-hydroperoxyoctadecadienoic acid
show the reaction diagram
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ir
linoleic acid + O2
?
show the reaction diagram
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?
linolenate + O2
(11R)-hydroperoxyoctadecadienoic acid + 13 S-hydroperoxyoctadecadienoic acid + 9(S/R)-hydroperoxyoctadecadienoic acid
show the reaction diagram
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Fo-MnLOX, with support of Ser348, binds linoleic acid so that the pro R rather than the pro S hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs
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?
linolenate + O2
(11S)-hydroperoxyoctadecadienoic acid + (13R)-hydroperoxyoctadecadienoic acid + (9S)-hydroperoxyoctadecadienoic acid
show the reaction diagram
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linolenate + O2 +
(11S)-hydroperoxyoctadecadienoic acid + (13R)-hydroperoxyoctadecadienoic acid + (9S)-hydroperoxyoctadecadienoic acid
show the reaction diagram
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?
linolenic acid + O2
11-(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid
show the reaction diagram
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?
lyso-phosphatidylcholine + O2
?
show the reaction diagram
additional information
?
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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
(9Z,11S,12Z)-11-hydroperoxyoctadeca-9,12-dienoate + O2
(9Z,11E, 13R)-13-hydroperoxyoctadeca-9,11-dienoate
show the reaction diagram
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-
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?
linoleate + O2
(9Z,11S,12Z)-11-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
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-
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?
linoleate + O2
(9Z,12Z)-(11S)-11-hydroperoxyoctadeca-9,12-dienoate
show the reaction diagram
B7JX99
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?
additional information
?
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13R-MnLOX forms (11S)-hydroperoxyoctadecadienoic acid and (11R)-hydroperoxyoctadecatrienoic acid as intermediates during the linear phase of oxidation and the (13R)-hydroperoxides as main end products
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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
Cu2+
enzyme-bound, is eliminated by denaturation of the enzyme protein
Fe3+
non-heme iron, required. The catalytic non-heme iron, deeply buried in the CspLOX2 active site, is coordinated by three invariant histidines (His257, His262, His449), Asn453 and the carboxy group of the C-terminal Ile569. The sixth ligand of the octahedrally coordinated iron, which is positioned towards the putative substrate binding channel, is a water (Fe2+-H2O) or hydroxide molecule (Fe3+-OH) acting as a catalytic base for hydrogen abstraction during catalysis, coordination of the iron cofactor, overview
Manganese
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mononuclear Mn center, 0.94 mol Mn per mol of protein
Mg2+
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activation less effective than with Ca2+ in phosphate buffer
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
arachidonic acid
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inhibits at 0.01 mM
eicosatetraynoic acid
N-(3-phenoxycinnamyl) acetohydroxamic acid
0.3 mM, complete inhibition
N-(3-phenoxycinnamyl)acetohydroxamic acid
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50% of control activity at 0.05 mM
Tween 20
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inhibition of Ca2+-activated enzyme at pH 7.5 and 6.4, no inhibition at pH 6.4 in the absence of Ca2+
additional information
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no inhibition by lipoxygenase inhibitors zileuton, esculetin and ICI 230487
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0106
(11Z,14Z)-eicosa-11,14-dienoic acid
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pH 9.0, temperature not specified in the publication, recombinant enzyme
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0.0112
(11Z,14Z,17Z)-eicosa-11,14,17-trienoic acid
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pH 9.0, temperature not specified in the publication, recombinant enzyme
0.01
(13R)-hydroperoxylinolenic acid
mutant enzyme G316A
0.0005 - 0.0073
(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid
0.0024 - 0.0071
alpha-linolenic acid
0.0024
linoleate
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pH 7.5, 18:3n-3 substrate
0.0044
linoleic acid
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0.0122
linolenate
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pH 9.0, temperature not specified in the publication, recombinant enzyme
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
40
alpha-linolenic acid
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40
linoleate
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pH 7.5, 18:3n-3 substrate
18.3
linoleic acid
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0.0122
linolenate
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pH 9.0, temperature not specified in the publication, recombinant enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.01
purified recombinant enzyme, pH 9.0, 21°C
18
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pH 9.0, 21°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.4
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in the absence of Ca2+
7
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broad pH optimum
7.2
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in the presence of 0.37 mM Ca2+
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 7
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in the absence of Ca2+
4 - 9
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in the presence of 0.37 mM Ca2+
5 - 11
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at pH 5.0 64% of maximum activity, at pH 11 69% of maximum activity
7
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93% of maximum activity
8
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98% of maximum activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12 - 60
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8.8% of maximal activity at 12°C and 100% of maximal activity at 60°C, activity decreases sharply at 63°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Magnaporthe oryzae (strain 70-15 / ATCC MYA-4617 / FGSC 8958)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
73000
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SDS-PAGE after deglycosylation
135000
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gel filtration with Superdex 200 in 6 M urea
160000
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gel filtration with Superdex 200
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
the deglycosylation process slightly decreases the melting temperature of recombinant Mo-MnLOX from 60°C to 56°C
63
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incubation for 5 min over 63°C decreases activity, no activity at 75°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
9S-LOX activity is less robust during expression and purification, possibly due to proteolysis, stability is not improved by addition of 0.001 mM pepstatin A, 0.001 mM leupeptin, or 1 mM EDTA to the growth medium
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 3 mM NaN3, 0.2 mM GSH, several months, no loss of activity
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4°C, stable for months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
phenyl-Sepharose column chromatography
recombinant His-tagged enzyme from Escherichia coli strain BL21 Star by nickel affinity chromatography, gel filtration, and ultrafiltration
recombinant mini 13R-MnLOX from Pichia pastoris by hydrophobic interaction chromatography and gel filtration
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recombinant secreted enzyme 43fold from Pichia pastoris by hydrophobic interaction chromatography and gel filtration
recombinant secreted wild-type and mutant enzymes from Pichia pastoris by hydrophobic interaction chromatography, ultrafiltration, and gel filtration
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recombinant wild-type and mutant enzymes from Pichia pastoris by hydrophobic interaction chromatography, dialfiltration, and gel filtration
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recombinant wild-type and mutant enzymes from Pichia pastoris cell medium by hydrophobic interaction and ion exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction, expression, and secretion of truncated 13R-MnLOX, from mini DNA coding for the sequence Glu-Phe-His6-Leu-Gln-Thr39-Val-Leu-Pro to Val618, in Pichia pastoris
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DNA and amino acid sequence determination and analysis, phylogenetic tree of two LOX prototypes from different species, recombinant expression and secretion of wild-type and mutant enzymes in Pichia pastoris
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expressed in Pichia pastoris
expression in Pichia pastoris
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expression of wild-type and G316A mutant enzymes in Pichia pastoris strain X-33, secretion to the cell medium
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gene MGG_08499, DNA and amino acid sequence determination and analysis, recombinant expression and secretion of the enzyme in Pichia pastoris
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 Star
recombinant expression of wild-type and mutant enzymes in Pichia pastoris
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A300G
site-directed mutagenesis, structure comparison with wild-type enzyme, enantioselectivity towards formation of 9R- and 13S-hydroperoxyoctadeca-9,12-dienoate is increased compared to the wild-type enzyme
I296A
site-directed mutagenesis, structure comparison with wild-type enzyme, the stereospecificity of the mutant activity is inverted compared to the wild-type enzyme
L258V
site-directed mutagenesis, structure comparison with wild-type enzyme, the mutation only slightly affects the enzyme activity
L502V
site-directed mutagenesis, structure comparison with wild-type enzyme, enantioselectivity towards formation of 9R- and 13S-hydroperoxyoctadeca-9,12-dienoate is increased compared to the wild-type enzyme
L506V
site-directed mutagenesis, structure comparison with wild-type enzyme, the stereospecificity of the mutant activity is inverted compared to the wild-type enzyme
L530R
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site-directed mutagenesis
S348F
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site-directed mutagenesis, the mutant enzyme shows altered reaction stereospecificity compared to the wild-type enzyme
F337I
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site-directed mutagenesis, replacement of Phe337 with Ile changes the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from 100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 is retained, suggesting antarafacial hydrogen abstraction and oxygenation. The mutant shows highly reduced activity compared to the wild-type enzyme
F347A
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site-directed mutagenesis, the mutant oxidizes octadeca-9,11-dienoic acid to 11-hydroperoxyoctadecadienoate/(13R)-hydroperoxyoctadecadienoate/(9S)-hydroperoxyoctadecadienoate in an initial ratio of 50/42/8 and with almost complete consumption of 11-hydroperoxyoctadecadienoate to a ratio of 2/84/14. The (9E,11Z,15E)-octadeca-9,11,15-trienoic acid is transformed to 11- and (13R)-hydroperoxyoctadecatrienoic acid and to traces of (9S)-hydroperoxyoctadecatrienoic acid, essentially as native 13R-MnLOX
F347L
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site-directed mutagenesis, (9E,11Z,15E)-octadeca-9,11,15-trienoic acid is oxidized to a 2:3 mixture of (9S)- and (13R)-dihydroxyoctadecatrienoic acid
F347V
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site-directed mutagenesis
G316T
catalytically inactive
G316V
catalytically inactive
G332A
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site-directed mutagenesis, replacement of Gly332 with the larger hydrophobic residue selectively augments dehydration of (9Z,11E,13R,15Z)-13-hydroperoxyoctadeca-9,12,15-trienoic acid and increases the oxidation at C-13 of 18:1n-6
H274Q
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no enzymic activity, loss of more than 95% of manganese content
H278E
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no enzymic activity, loss of more than 95% of manganese content
H462E
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no enzymic activity, loss of more than 95% of manganese content
H463Q
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no significant change in activity
K52N
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site-directed mutagenesis
L176K
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site-directed mutagenesis
L336A
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site-directed mutagenesis, replacement of Leu336 with the smaller hydrophobic residue shifts the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3, the mutant shows highly reduced activity compared to the wild-type enzyme
L336F
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site-directed mutagenesis, replacement of Leu336 with the larger hydrophobic residue selectively augments dehydration of (9Z,11E,13R,15Z)-13-hydroperoxyoctadeca-9,12,15-trienoic acid and increases the oxidation at C-13 of 18:1n-6, the mutant shows highly reduced activity compared to the wild-type enzyme
L336G
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site-directed mutagenesis, replacement of Leu336 with the smaller hydrophobic residue shifts the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3, the mutant shows highly reduced activity compared to the wild-type enzyme
L336V
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site-directed mutagenesis, replacement of Leu336 with the smaller hydrophobic residue shifts the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3, the mutant shows highly reduced activity compared to the wild-type enzyme
N466Q
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no significant change in activity
Q467N
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no significant change in activity
S469A
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site-directed mutagenesis, the mutation hardly affects the bis-allylic hydroperoxide rearrangement, the mutant shows the same substrate profile as the wild-type enzyme
Y158C
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site-directed mutagenesis
additional information