Information on EC 5.4.4.6 - 9,12-octadecadienoate 8-hydroperoxide 8S-isomerase

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The expected taxonomic range for this enzyme is: Gaeumannomyces graminis

EC NUMBER
COMMENTARY hide
5.4.4.6
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RECOMMENDED NAME
GeneOntology No.
9,12-octadecadienoate 8-hydroperoxide 8S-isomerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate = (7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-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
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate hydroxymutase [(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate-forming]
The enzyme contains heme. The bifunctional enzyme from Gaeumannomyces graminis catalyses the oxidation of linoleic acid to (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate (cf. EC 1.13.11.60, linoleate 8R-lipoxygenase), which is then isomerized to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate [3].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(8R,9Z,12Z)-8-hydroperoxy-10,12-octadecadienoate
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
show the reaction diagram
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
(7S,8S,9Z,12Z)-7,8-dihydroxy-9,12-octadecadienoate
show the reaction diagram
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
show the reaction diagram
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conversion of (8R)-hydroperoxylinoleic acid into (7S,8S)-dihydroxylinoleic acid by stereospecific elimination of the pro-S hydrogen from C-7 and intramolecular suprafacial insertion of oxygen at C-7
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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
(8R,9Z,12Z)-8-hydroperoxy-10,12-octadecadienoate
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
show the reaction diagram
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
(7S,8S,9Z,12Z)-7,8-dihydroxy-9,12-octadecadienoate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
diethyldicarbonate
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Tetranitromethane
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0.4 mM, 50% inhibition
additional information
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catalysis declines due to suicide inactivation; catalysis declines due to suicide inactivation. Following column chromatography, e.g. ion exchange or zinc-affinity chromatography, hydroperoxide isomerase activity is often decreased and sometimes even undetectable by TLC, whereas 8-dioxygenase activity is always present. However, storage of the enzyme for a few days on ice often restores the hydroperoxide isomerase and appeares to increase the LDS activity as well
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
CHAPS
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0.5 mM, augments enzyme activity
Tween 20
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0.04%, augments enzyme activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10
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broad optimum
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
108000
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4 * 108000, calculation from sequence
130000
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4 * 130000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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; 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
Purification/COMMENTARY
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 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; 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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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H203Q
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weak activity
H379Q
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inactive mutant enzyme
K540L
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inactive mutant enzyme
K540Q
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inactive mutant enzyme
K540R
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inactive mutant enzyme
N216Q
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inactive mutant enzyme
Y329F
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weak activity
Y329L
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inactive mutant enzyme
Y376F
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inactive mutant enzyme
Y378F
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inactive mutant enzyme
Y378S
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inactive mutant enzyme
Y531F
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inactive mutant enzyme
additional information
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treatment with alpha-mannosidase to shorten N- and O-linked mannosides inhibis the hydroperoxide isomerase but not the 8(R)-dioxygenase; treatment with alpha-mannosidase to shorten N- and O-linked mannosides inhibis the hydroperoxide isomerase but not the 8(R)-dioxygenase