BRENDA - Enzyme Database show

Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate

Seo, M.J.; Shin, K.C.; An, J.U.; Kang, W.R.; Ko, Y.J.; Oh, D.K.; Appl. Microbiol. Biotechnol. 100, 3087-3099 (2016)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
1.13.11.60
DMSO
the whole enzyme activity is the highest using DMSO among the organic solvents tested, and its optimal concentration is 2.5% v/v
Colletotrichum gloeosporioides
Cloned(Commentary)
EC Number
Commentary
Organism
1.13.11.60
expression in Escherichia coli; phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566
Colletotrichum gloeosporioides
5.4.4.6
phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566
Colletotrichum gloeosporioides
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.13.11.60
A891G
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
C1038S
site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
H1036A
site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
H1036A/C1038S
site-directed mutagenesis, the double mutant converts linoleic acid to only 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
additional information
comparison of substrate specificities of wild-type an d mutant enzymes, overview
Colletotrichum gloeosporioides
1.13.11.60
N895D
site-directed mutagenesis, the mutant produces a reduced amount of 8-HPODE, compared to wild-type, without the accumulation of DiHODE
Colletotrichum gloeosporioides
1.13.11.60
N895L
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
N895Q
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
Q898E
site-directed mutagenesis, the mutant converts linoleic acid to 8,11-DiHODE as major product and 7,8-DiHODE as minor product
Colletotrichum gloeosporioides
1.13.11.60
Q898L
site-directed mutagenesis, the mutant converts linoleic acid to 8,11-DiHODE as major product and 7,8-DiHODE as minor product
Colletotrichum gloeosporioides
1.13.11.60
Y380F
site-directed mutagenesis, the mutant enzyme produces trace amount of 7,8-DiHODE without the accumulation of 8-HPODE when incubated with linoleic acid as substrate
Colletotrichum gloeosporioides
5.4.4.6
A891G
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
C1038S
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
H1036A
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
H1036A/C1038S
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
additional information
comparison of substrate specificities of wild-type and mutant enzymes, overview
Colletotrichum gloeosporioides
5.4.4.6
N895D
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
N895L
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
N895Q
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Q898E
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Q898L
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Y380F
site-directed mutagenesis
Colletotrichum gloeosporioides
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.13.11.60
0.0193
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0193
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.022
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0264
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0293
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0305
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0319
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0319
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0324
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0324
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0333
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0358
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0358
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0368
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0369
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0371
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.048
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0223
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0333
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0371
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.048
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.13.11.60
510000
-
gel filtration; recombinant enzyme, gel filtration
Colletotrichum gloeosporioides
5.4.4.6
510000
-
recombinant enzyme, gel filtration
Colletotrichum gloeosporioides
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.13.11.60
linoleate + O2
Colletotrichum gloeosporioides
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
-
?
5.4.4.6
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
Colletotrichum gloeosporioides
-
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
-
-
r
Organic Solvent Stability
EC Number
Organic Solvent
Commentary
Organism
1.13.11.60
DMSO
maximum activity in presence of 2.5% DMSO
Colletotrichum gloeosporioides
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.13.11.60
Colletotrichum gloeosporioides
A0A0S2SWE4
bifunctional enzyme, the second domain isomerizes the product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate, cf. EC 5.4.4.6; i.e. Colletotrichum gloeosporioides
-
1.13.11.60
Colletotrichum gloeosporioides KACC 40961
A0A0S2SWE4
i.e. Colletotrichum gloeosporioides
-
5.4.4.6
Colletotrichum gloeosporioides
A0A0S2SWE4
i.e. Colletotrichum gloeosporioides
-
5.4.4.6
Colletotrichum gloeosporioides KACC 40961
A0A0S2SWE4
i.e. Colletotrichum gloeosporioides
-
Purification (Commentary)
EC Number
Commentary
Organism
1.13.11.60
recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography
Colletotrichum gloeosporioides
5.4.4.6
recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography
Colletotrichum gloeosporioides
Source Tissue
EC Number
Source Tissue
Commentary
Organism
Textmining
1.13.11.60
mycelium
-
Colletotrichum gloeosporioides
-
5.4.4.6
mycelium
-
Colletotrichum gloeosporioides
-
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.13.11.60
0.39
-
purified recombinant enzyme, whole enzyme activity, substrate palmitoleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.48
-
purified recombinant enzyme, whole enzyme activity, substrate oleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.75
-
purified recombinant enzyme, whole enzyme activity, substrate alpha-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.87
-
pH 6.5, 40°C; purified recombinant enzyme, whole enzyme activity, substrate (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.17
-
purified recombinant enzyme, N-terminal domain activity, substrate arachidate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.18
-
purified recombinant enzyme, N-terminal domain activity, substrate dihomo-gamma-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.2
-
purified recombinant enzyme, N-terminal domain activity, substrate eicosadienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.39
-
purified recombinant enzyme, N-terminal domain activity, substrate eicosenoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.39
-
purified recombinant enzyme, whole enzyme activity, substrate palmitoleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.48
-
purified recombinant enzyme, whole enzyme activity, substrate oleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.75
-
purified recombinant enzyme, whole enzyme activity, substrate alpha-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.87
-
purified recombinant enzyme, whole enzyme activity, substrate (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.13.11.60
alpha-linolenate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxy-9,12,15-octadecatrienoate
-
-
-
?
1.13.11.60
alpha-linolenate + O2
via (9Z,12Z,15Z)-8-hydroperoxyoctadeca-9,12,15-trienoic acid, best substrate with respect to whole enzyme activity
741712
Colletotrichum gloeosporioides
(9Z,12Z,15Z)-7,8-dihydroxyoctadeca-9,12,15-trienoic acid
-
-
-
?
1.13.11.60
arachidonate + O2
-
741712
Colletotrichum gloeosporioides
(5Z,8Z,11Z,14Z)-8-hydroxyeicosa-5,8,11,14-tetraenoic acid
-
-
-
?
1.13.11.60
arachidonic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyarachidonic acid
-
-
-
?
1.13.11.60
dihomo-gamma-linolenate + O2
-
741712
Colletotrichum gloeosporioides
(8Z,11Z,12Z)-8-hydroxyeicosa-8,11,12-trienoic acid
-
-
-
?
1.13.11.60
dihomo-gamma-linolenic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxydihomo-gamma-linolenic acid
-
-
-
?
1.13.11.60
eicosadienoate + O2
-
741712
Colletotrichum gloeosporioides
(11Z,14Z)-8-hydroxyeicosa-11,14-dienoic acid
-
-
-
?
1.13.11.60
eicosadienoic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyeicosadienoic acid
-
-
-
?
1.13.11.60
eicosenoate + O2
-
741712
Colletotrichum gloeosporioides
(11Z)-8-hydroxyeicosa-11-enoic acid
-
-
-
?
1.13.11.60
eicosenoic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyeicosenoic acid
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(7S,8S)-7,8-dihydroxy-(9Z,12Z)-octadecadienoic acid
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(R)-8-hydroperoxy-(9Z,12Z)-octadecadienoic acid
-
-
-
?
1.13.11.60
additional information
bifunctional enzyme, the second domain isomerizes the product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate, cf. EC 5.4.4.6
741712
Colletotrichum gloeosporioides
?
-
-
-
-
1.13.11.60
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (9Z,12Z)-7S,8S-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5R,8R,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides
?
-
-
-
-
1.13.11.60
additional information
bifunctional enzyme, the second domain isomerizes the product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate, cf. EC 5.4.4.6
741712
Colletotrichum gloeosporioides KACC 40961
?
-
-
-
-
1.13.11.60
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (9Z,12Z)-7S,8S-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5R,8R,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides KACC 40961
?
-
-
-
-
1.13.11.60
oleate + O2
-
741712
Colletotrichum gloeosporioides
(9Z)-8-hydroperoxyoctadec-9-enoate
-
-
-
?
1.13.11.60
oleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(9Z)-8-hydroperoxyoctadec-9-enoate
-
-
-
?
1.13.11.60
oleate + O2
via (9Z)-8-hydroperoxyoctadeca-9-enoic acid
741712
Colletotrichum gloeosporioides
(9Z)-7,8-dihydroxyoctadeca-9-enoic acid
-
-
-
?
1.13.11.60
palmitoleate + O2
-
741712
Colletotrichum gloeosporioides
(9Z)-8-hydroperoxyhexadec-9-enoate
-
-
-
?
1.13.11.60
palmitoleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(9Z)-8-hydroperoxyhexadec-9-enoate
-
-
-
?
1.13.11.60
palmitoleate + O2
via (9Z)-dihydroperoxyhexadeca-9-enoate
741712
Colletotrichum gloeosporioides
(9Z)-5,8-dihydroxyhexadeca-9-enoic acid
-
-
-
?
5.4.4.6
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
741712
Colletotrichum gloeosporioides
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
-
-
-
r
5.4.4.6
alpha-linolenate + O2
via (9Z,12Z,15Z)-8-hydroperoxyoctadeca-9,12,15-trienoic acid, best substrate with respect to whole enzyme activity
741712
Colletotrichum gloeosporioides
(9Z,12Z,15Z)-7,8-dihydroperoxyoctadeca-9,12,15-trienoic acid
-
-
-
?
5.4.4.6
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (7S,8S,9Z,12Z)-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5S,8R,9Z,12Z)-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides
?
-
-
-
-
5.4.4.6
oleate + O2
via (9Z)-8-hydroperoxyoctadeca-9-enoic acid
741712
Colletotrichum gloeosporioides
(9Z)-7,8-dihydroxyoctadeca-9-enoic acid
-
-
-
?
5.4.4.6
palmitoleate + O2
via (9Z)-8-dihydroperoxyhexadeca-9-enoate
741712
Colletotrichum gloeosporioides
(9Z)-5,8-dihydroxyhexadeca-9-enoic acid
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.13.11.60
homotetramer
4 * 127000, recombinant His-tagged enzyme, SDS-PAGE, 4 * 127413, sequence calculation
Colletotrichum gloeosporioides
1.13.11.60
More
the enzyme diol synthase is a fusion protein, consisting of an N-terminal dioxygenase domain and a C-terminal cytochrome P450/hydroperoxide isomerase domain
Colletotrichum gloeosporioides
1.13.11.60
trimer
3 * 127000, SDS-PAGE, 3 * 127413, calculated
Colletotrichum gloeosporioides
5.4.4.6
homotetramer
4 * 127000, recombinant His-tagged enzyme, SDS-PAGE, 4 * 127413, sequence calculation
Colletotrichum gloeosporioides
5.4.4.6
More
the enzyme diol synthase is a fusion protein, consisting of an N-terminal dioxygenase domain and a C-terminal cytochrome P450/hydroperoxide isomerase domain
Colletotrichum gloeosporioides
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.13.11.60
20
-
maximum of 8-diogenase reaction; N-terminal domain activity
Colletotrichum gloeosporioides
1.13.11.60
40
-
maximum of whole enzyme; whole enzyme activity
Colletotrichum gloeosporioides
5.4.4.6
40
-
whole enzyme activity
Colletotrichum gloeosporioides
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.13.11.60
20
50
purified recombinant enzyme, 2 h, the activities of whole 7,8-LDS and 8-dioxygenase in the N-terminal domain decrease as temperature increases. The N-terminal domain demonstrates a greater thermal stability than the C-terminal domain. The whole enzyme activity presentes 72% of the initial activity at 40°C after 2 h, while 8-dioxygenase activity in the N-terminal domain is 90%
Colletotrichum gloeosporioides
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.13.11.60
4.86
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
5.62
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
6.58
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
10.5
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.2
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.2
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.7
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.7
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
18.2
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
18.2
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
21.2
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
22
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
22
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
24.7
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
25.1
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
25.2
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
35
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
35
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
4.86
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
5.62
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
6.58
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
10.5
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.13.11.60
additional information
-
the maximum activity for the conversion of linoleic acid to (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) by 8-dioxygenase in the N-terminal domain of 7,8-LDS is observed at pH 7.0 and 20°C, whereas that for the conversion of linoleic acid to (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by the whole enzyme is observed at pH 6.5 and 40°C
Colletotrichum gloeosporioides
1.13.11.60
6.5
-
maximum of bifunctional enzyme; whole enzyme activity
Colletotrichum gloeosporioides
1.13.11.60
7
-
maximum of 8-diogenase reaction; N-terminal domain activity
Colletotrichum gloeosporioides
5.4.4.6
additional information
-
the maximum activity for the conversion of linoleic acid to (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) by 8-dioxygenase in the N-terminal domain of 7,8-LDS is observed at pH 7.0 and 20°C, whereas that for the conversion of linoleic acid to (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by the whole enzyme is observed at pH 6.5 and 40°C
Colletotrichum gloeosporioides
5.4.4.6
6.5
-
whole enzyme activity
Colletotrichum gloeosporioides
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
1.13.11.60
DMSO
the whole enzyme activity is the highest using DMSO among the organic solvents tested, and its optimal concentration is 2.5% v/v
Colletotrichum gloeosporioides
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.13.11.60
expression in Escherichia coli; phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566
Colletotrichum gloeosporioides
5.4.4.6
phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566
Colletotrichum gloeosporioides
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.13.11.60
A891G
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
C1038S
site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
H1036A
site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
H1036A/C1038S
site-directed mutagenesis, the double mutant converts linoleic acid to only 8-HPODE
Colletotrichum gloeosporioides
1.13.11.60
additional information
comparison of substrate specificities of wild-type an d mutant enzymes, overview
Colletotrichum gloeosporioides
1.13.11.60
N895D
site-directed mutagenesis, the mutant produces a reduced amount of 8-HPODE, compared to wild-type, without the accumulation of DiHODE
Colletotrichum gloeosporioides
1.13.11.60
N895L
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
N895Q
site-directed mutagenesis, the mutant converts linoleic acid to 7,8-DiHODE as major product and 8,11-DiHODE as minor product, which is the similar pattern as the wild-type enzyme
Colletotrichum gloeosporioides
1.13.11.60
Q898E
site-directed mutagenesis, the mutant converts linoleic acid to 8,11-DiHODE as major product and 7,8-DiHODE as minor product
Colletotrichum gloeosporioides
1.13.11.60
Q898L
site-directed mutagenesis, the mutant converts linoleic acid to 8,11-DiHODE as major product and 7,8-DiHODE as minor product
Colletotrichum gloeosporioides
1.13.11.60
Y380F
site-directed mutagenesis, the mutant enzyme produces trace amount of 7,8-DiHODE without the accumulation of 8-HPODE when incubated with linoleic acid as substrate
Colletotrichum gloeosporioides
5.4.4.6
A891G
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
C1038S
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
H1036A
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
H1036A/C1038S
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
additional information
comparison of substrate specificities of wild-type and mutant enzymes, overview
Colletotrichum gloeosporioides
5.4.4.6
N895D
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
N895L
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
N895Q
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Q898E
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Q898L
site-directed mutagenesis
Colletotrichum gloeosporioides
5.4.4.6
Y380F
site-directed mutagenesis
Colletotrichum gloeosporioides
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.13.11.60
0.0193
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0193
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.022
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0264
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0293
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0305
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0319
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0319
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0324
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0324
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0333
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0358
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0358
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0368
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0369
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.0371
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.048
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0223
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0333
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.0371
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.048
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.13.11.60
510000
-
gel filtration; recombinant enzyme, gel filtration
Colletotrichum gloeosporioides
5.4.4.6
510000
-
recombinant enzyme, gel filtration
Colletotrichum gloeosporioides
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.13.11.60
linoleate + O2
Colletotrichum gloeosporioides
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
-
?
5.4.4.6
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
Colletotrichum gloeosporioides
-
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
-
-
r
Organic Solvent Stability (protein specific)
EC Number
Organic Solvent
Commentary
Organism
1.13.11.60
DMSO
maximum activity in presence of 2.5% DMSO
Colletotrichum gloeosporioides
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.13.11.60
recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography
Colletotrichum gloeosporioides
5.4.4.6
recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography
Colletotrichum gloeosporioides
Source Tissue (protein specific)
EC Number
Source Tissue
Commentary
Organism
Textmining
1.13.11.60
mycelium
-
Colletotrichum gloeosporioides
-
5.4.4.6
mycelium
-
Colletotrichum gloeosporioides
-
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.13.11.60
0.39
-
purified recombinant enzyme, whole enzyme activity, substrate palmitoleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.48
-
purified recombinant enzyme, whole enzyme activity, substrate oleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.75
-
purified recombinant enzyme, whole enzyme activity, substrate alpha-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
0.87
-
pH 6.5, 40°C; purified recombinant enzyme, whole enzyme activity, substrate (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.17
-
purified recombinant enzyme, N-terminal domain activity, substrate arachidate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.18
-
purified recombinant enzyme, N-terminal domain activity, substrate dihomo-gamma-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.2
-
purified recombinant enzyme, N-terminal domain activity, substrate eicosadienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1.39
-
purified recombinant enzyme, N-terminal domain activity, substrate eicosenoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.39
-
purified recombinant enzyme, whole enzyme activity, substrate palmitoleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.48
-
purified recombinant enzyme, whole enzyme activity, substrate oleate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.75
-
purified recombinant enzyme, whole enzyme activity, substrate alpha-linolenate, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
0.87
-
purified recombinant enzyme, whole enzyme activity, substrate (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate, pH 6.5, 40°C
Colletotrichum gloeosporioides
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.13.11.60
alpha-linolenate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxy-9,12,15-octadecatrienoate
-
-
-
?
1.13.11.60
alpha-linolenate + O2
via (9Z,12Z,15Z)-8-hydroperoxyoctadeca-9,12,15-trienoic acid, best substrate with respect to whole enzyme activity
741712
Colletotrichum gloeosporioides
(9Z,12Z,15Z)-7,8-dihydroxyoctadeca-9,12,15-trienoic acid
-
-
-
?
1.13.11.60
arachidonate + O2
-
741712
Colletotrichum gloeosporioides
(5Z,8Z,11Z,14Z)-8-hydroxyeicosa-5,8,11,14-tetraenoic acid
-
-
-
?
1.13.11.60
arachidonic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyarachidonic acid
-
-
-
?
1.13.11.60
dihomo-gamma-linolenate + O2
-
741712
Colletotrichum gloeosporioides
(8Z,11Z,12Z)-8-hydroxyeicosa-8,11,12-trienoic acid
-
-
-
?
1.13.11.60
dihomo-gamma-linolenic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxydihomo-gamma-linolenic acid
-
-
-
?
1.13.11.60
eicosadienoate + O2
-
741712
Colletotrichum gloeosporioides
(11Z,14Z)-8-hydroxyeicosa-11,14-dienoic acid
-
-
-
?
1.13.11.60
eicosadienoic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyeicosadienoic acid
-
-
-
?
1.13.11.60
eicosenoate + O2
-
741712
Colletotrichum gloeosporioides
(11Z)-8-hydroxyeicosa-11-enoic acid
-
-
-
?
1.13.11.60
eicosenoic acid + O2
-
741712
Colletotrichum gloeosporioides
8-hydroxyeicosenoic acid
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(7S,8S)-7,8-dihydroxy-(9Z,12Z)-octadecadienoic acid
-
-
-
?
1.13.11.60
linoleate + O2
-
741712
Colletotrichum gloeosporioides
(R)-8-hydroperoxy-(9Z,12Z)-octadecadienoic acid
-
-
-
?
1.13.11.60
additional information
bifunctional enzyme, the second domain isomerizes the product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate, cf. EC 5.4.4.6
741712
Colletotrichum gloeosporioides
?
-
-
-
-
1.13.11.60
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (9Z,12Z)-7S,8S-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5R,8R,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides
?
-
-
-
-
1.13.11.60
additional information
bifunctional enzyme, the second domain isomerizes the product (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate to (7S,8S,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoate, cf. EC 5.4.4.6
741712
Colletotrichum gloeosporioides KACC 40961
?
-
-
-
-
1.13.11.60
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (9Z,12Z)-7S,8S-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5R,8R,9Z,12Z)-5,8-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides KACC 40961
?
-
-
-
-
1.13.11.60
oleate + O2
-
741712
Colletotrichum gloeosporioides
(9Z)-8-hydroperoxyoctadec-9-enoate
-
-
-
?
1.13.11.60
oleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(9Z)-8-hydroperoxyoctadec-9-enoate
-
-
-
?
1.13.11.60
oleate + O2
via (9Z)-8-hydroperoxyoctadeca-9-enoic acid
741712
Colletotrichum gloeosporioides
(9Z)-7,8-dihydroxyoctadeca-9-enoic acid
-
-
-
?
1.13.11.60
palmitoleate + O2
-
741712
Colletotrichum gloeosporioides
(9Z)-8-hydroperoxyhexadec-9-enoate
-
-
-
?
1.13.11.60
palmitoleate + O2
-
741712
Colletotrichum gloeosporioides KACC 40961
(9Z)-8-hydroperoxyhexadec-9-enoate
-
-
-
?
1.13.11.60
palmitoleate + O2
via (9Z)-dihydroperoxyhexadeca-9-enoate
741712
Colletotrichum gloeosporioides
(9Z)-5,8-dihydroxyhexadeca-9-enoic acid
-
-
-
?
5.4.4.6
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
-
741712
Colletotrichum gloeosporioides
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate
-
-
-
r
5.4.4.6
alpha-linolenate + O2
via (9Z,12Z,15Z)-8-hydroperoxyoctadeca-9,12,15-trienoic acid, best substrate with respect to whole enzyme activity
741712
Colletotrichum gloeosporioides
(9Z,12Z,15Z)-7,8-dihydroperoxyoctadeca-9,12,15-trienoic acid
-
-
-
?
5.4.4.6
additional information
the bifunctional enzyme converts linoleic acid to a product, identified as (9Z,12Z)7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (kcat/Km) of 7,8-diol synthase for the conversion of fatty acid to dihydroxy fatty acid follows the descending order linoleic acid, alpha-linolenic acid, oleic acid, and palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). Substrate specificity, overview. The reaction via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate and the accumulation of 8-HPODE is due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain, EC 5.4.4.6. 8-HPODE is subsequently isomerized to (7S,8S,9Z,12Z)-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) or (5S,8R,9Z,12Z)-dihydroxyoctadeca-9,12-dienoic acid (5,8-DiHODE) by the C-terminal hydroperoxide isomerase domain of diol synthase. No activity with conjugated linoleic acid, docosapentaenoic acid, and docosahexaenoic acid, and no isomerase activity and no whole enzyme acitivity, but dioxygenase activity with eicosenoic acid, eicosadienoic acid, dihomo-gamma-linolenic acid, and arachidonic acid
741712
Colletotrichum gloeosporioides
?
-
-
-
-
5.4.4.6
oleate + O2
via (9Z)-8-hydroperoxyoctadeca-9-enoic acid
741712
Colletotrichum gloeosporioides
(9Z)-7,8-dihydroxyoctadeca-9-enoic acid
-
-
-
?
5.4.4.6
palmitoleate + O2
via (9Z)-8-dihydroperoxyhexadeca-9-enoate
741712
Colletotrichum gloeosporioides
(9Z)-5,8-dihydroxyhexadeca-9-enoic acid
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.13.11.60
homotetramer
4 * 127000, recombinant His-tagged enzyme, SDS-PAGE, 4 * 127413, sequence calculation
Colletotrichum gloeosporioides
1.13.11.60
More
the enzyme diol synthase is a fusion protein, consisting of an N-terminal dioxygenase domain and a C-terminal cytochrome P450/hydroperoxide isomerase domain
Colletotrichum gloeosporioides
1.13.11.60
trimer
3 * 127000, SDS-PAGE, 3 * 127413, calculated
Colletotrichum gloeosporioides
5.4.4.6
homotetramer
4 * 127000, recombinant His-tagged enzyme, SDS-PAGE, 4 * 127413, sequence calculation
Colletotrichum gloeosporioides
5.4.4.6
More
the enzyme diol synthase is a fusion protein, consisting of an N-terminal dioxygenase domain and a C-terminal cytochrome P450/hydroperoxide isomerase domain
Colletotrichum gloeosporioides
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.13.11.60
20
-
maximum of 8-diogenase reaction; N-terminal domain activity
Colletotrichum gloeosporioides
1.13.11.60
40
-
maximum of whole enzyme; whole enzyme activity
Colletotrichum gloeosporioides
5.4.4.6
40
-
whole enzyme activity
Colletotrichum gloeosporioides
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.13.11.60
20
50
purified recombinant enzyme, 2 h, the activities of whole 7,8-LDS and 8-dioxygenase in the N-terminal domain decrease as temperature increases. The N-terminal domain demonstrates a greater thermal stability than the C-terminal domain. The whole enzyme activity presentes 72% of the initial activity at 40°C after 2 h, while 8-dioxygenase activity in the N-terminal domain is 90%
Colletotrichum gloeosporioides
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.13.11.60
4.86
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
5.62
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
6.58
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
10.5
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.2
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.2
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.7
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
17.7
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
18.2
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
18.2
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
21.2
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
22
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
22
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
24.7
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C; recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
25.1
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
25.2
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
35
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
35
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
4.86
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
5.62
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
6.58
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
10.5
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.13.11.60
additional information
-
the maximum activity for the conversion of linoleic acid to (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) by 8-dioxygenase in the N-terminal domain of 7,8-LDS is observed at pH 7.0 and 20°C, whereas that for the conversion of linoleic acid to (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by the whole enzyme is observed at pH 6.5 and 40°C
Colletotrichum gloeosporioides
1.13.11.60
6.5
-
maximum of bifunctional enzyme; whole enzyme activity
Colletotrichum gloeosporioides
1.13.11.60
7
-
maximum of 8-diogenase reaction; N-terminal domain activity
Colletotrichum gloeosporioides
5.4.4.6
additional information
-
the maximum activity for the conversion of linoleic acid to (9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic acid (8-HPODE) by 8-dioxygenase in the N-terminal domain of 7,8-LDS is observed at pH 7.0 and 20°C, whereas that for the conversion of linoleic acid to (9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoic acid (7,8-DiHODE) by the whole enzyme is observed at pH 6.5 and 40°C
Colletotrichum gloeosporioides
5.4.4.6
6.5
-
whole enzyme activity
Colletotrichum gloeosporioides
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.13.11.60
100
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
170
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
340
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
370
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
466.1
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
470
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
508.4
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
510
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
554.9
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
560
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
679
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
680
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
690
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
695.1
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
840
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
843
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
950
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
954.54
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1813.5
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1820
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
8.125
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
14.41
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
23.45
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
33.62
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.13.11.60
100
-
palmitoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
170
-
oleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
340
-
alpha-linolenate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
370
-
linoleate
bifunctional enzyme, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
466.1
-
dihomo-gamma-linolenate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
470
-
dihomo-gamma-linolenic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
508.4
-
arachidonate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
510
-
arachidonic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
554.9
-
eicosadienoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
560
-
eicosadienoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
679
-
eicosenoate
recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
680
-
eicosenoic acid
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
690
-
palmitoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
695.1
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
840
-
oleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
843
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
950
-
alpha-linolenate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
954.54
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1813.5
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
1.13.11.60
1820
-
linoleate
N-terminal 8-dioxygenase domain, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
8.125
-
palmitoleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
14.41
-
oleate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
23.45
-
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides
5.4.4.6
33.62
-
alpha-linolenate
recombinant enzyme, whole enzyme activity, pH 6.5, 40°C
Colletotrichum gloeosporioides