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Literature summary extracted from
- Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate (2016), Appl. Microbiol. Biotechnol., 100, 3087-3099 .
Activating Compound
| EC Number | Activating Compound | Comment | 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 | Cloned (Comment) | Organism |
|---|---|---|
| 1.13.11.60 | expression in Escherichia coli | Colletotrichum gloeosporioides |
| 1.13.11.60 | 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 |
Protein Variants
| EC Number | Protein Variants | Comment | 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 | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.13.11.60 | 0.0193 | - |
linoleate | N-terminal 8-dioxygenase domain, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 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 | 0.022 | - |
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 | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 0.0264 | - |
alpha-linolenate | 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 | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 0.0293 | - |
oleate | 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 | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 0.0305 | - |
palmitoleate | recombinant enzyme, whole enzyme 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 | 0.0319 | - |
eicosadienoate | 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 | 0.0324 | - |
eicosenoate | recombinant enzyme, N-terminal domain activity, 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 | - |
arachidonic acid | N-terminal 8-dioxygenase domain, 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 | 0.0368 | - |
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] | Comment | Organism |
|---|---|---|---|---|
| 1.13.11.60 | 510000 | - |
gel filtration | Colletotrichum gloeosporioides |
| 1.13.11.60 | 510000 | - |
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 | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 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 | Comment | Organism |
|---|---|---|---|
| 1.13.11.60 | DMSO | maximum activity in presence of 2.5% DMSO | Colletotrichum gloeosporioides |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.13.11.60 | Colletotrichum gloeosporioides | A0A0S2SWE4 | i.e. Colletotrichum gloeosporioides | - |
| 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 | - |
| 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 | Purification (Comment) | 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 | Comment | 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] | Comment | 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 | Colletotrichum gloeosporioides |
| 1.13.11.60 | 0.87 | - |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.13.11.60 | alpha-linolenate + O2 | - |
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 | Colletotrichum gloeosporioides | (9Z,12Z,15Z)-7,8-dihydroxyoctadeca-9,12,15-trienoic acid | - |
? | |
| 1.13.11.60 | arachidonate + O2 | - |
Colletotrichum gloeosporioides | (5Z,8Z,11Z,14Z)-8-hydroxyeicosa-5,8,11,14-tetraenoic acid | - |
? | |
| 1.13.11.60 | arachidonic acid + O2 | - |
Colletotrichum gloeosporioides | 8-hydroxyarachidonic acid | - |
? | |
| 1.13.11.60 | dihomo-gamma-linolenate + O2 | - |
Colletotrichum gloeosporioides | (8Z,11Z,14Z)-8-hydroxyeicosa-8,11,14-trienoic acid | - |
? | |
| 1.13.11.60 | dihomo-gamma-linolenic acid + O2 | - |
Colletotrichum gloeosporioides | 8-hydroxydihomo-gamma-linolenic acid | - |
? | |
| 1.13.11.60 | eicosadienoate + O2 | - |
Colletotrichum gloeosporioides | (11Z,14Z)-8-hydroxyeicosa-11,14-dienoic acid | - |
? | |
| 1.13.11.60 | eicosadienoic acid + O2 | - |
Colletotrichum gloeosporioides | 8-hydroxyeicosadienoic acid | - |
? | |
| 1.13.11.60 | eicosenoate + O2 | - |
Colletotrichum gloeosporioides | (11Z)-8-hydroxyeicosa-11-enoic acid | - |
? | |
| 1.13.11.60 | eicosenoic acid + O2 | - |
Colletotrichum gloeosporioides | 8-hydroxyeicosenoic acid | - |
? | |
| 1.13.11.60 | linoleate + O2 | - |
Colletotrichum gloeosporioides | (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate | - |
? | |
| 1.13.11.60 | linoleate + O2 | - |
Colletotrichum gloeosporioides KACC 40961 | (8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate | - |
? | |
| 1.13.11.60 | linoleate + O2 | - |
Colletotrichum gloeosporioides | (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | - |
? | |
| 1.13.11.60 | linoleate + O2 | - |
Colletotrichum gloeosporioides | (7S,8S)-7,8-dihydroxy-(9Z,12Z)-octadecadienoic acid | - |
? | |
| 1.13.11.60 | linoleate + O2 | - |
Colletotrichum gloeosporioides | (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoic 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 | 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 | 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 | 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 | Colletotrichum gloeosporioides KACC 40961 | ? | - |
? | |
| 1.13.11.60 | oleate + O2 | - |
Colletotrichum gloeosporioides | (9Z)-8-hydroperoxyoctadec-9-enoate | - |
? | |
| 1.13.11.60 | oleate + O2 | - |
Colletotrichum gloeosporioides KACC 40961 | (9Z)-8-hydroperoxyoctadec-9-enoate | - |
? | |
| 1.13.11.60 | oleate + O2 | via (9Z)-8-hydroperoxyoctadeca-9-enoic acid | Colletotrichum gloeosporioides | (9Z)-7,8-dihydroxyoctadeca-9-enoic acid | - |
? | |
| 1.13.11.60 | palmitoleate + O2 | - |
Colletotrichum gloeosporioides | (9Z)-8-hydroperoxyhexadec-9-enoate | - |
? | |
| 1.13.11.60 | palmitoleate + O2 | - |
Colletotrichum gloeosporioides KACC 40961 | (9Z)-8-hydroperoxyhexadec-9-enoate | - |
? | |
| 1.13.11.60 | palmitoleate + O2 | via (9Z)-dihydroperoxyhexadeca-9-enoate | Colletotrichum gloeosporioides | (9Z)-5,8-dihydroxyhexadeca-9-enoic acid | - |
? | |
| 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 | |
| 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 | 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 | Colletotrichum gloeosporioides | ? | - |
? | |
| 5.4.4.6 | oleate + O2 | via (9Z)-8-hydroperoxyoctadeca-9-enoic acid | Colletotrichum gloeosporioides | (9Z)-7,8-dihydroxyoctadeca-9-enoic acid | - |
? | |
| 5.4.4.6 | palmitoleate + O2 | via (9Z)-8-dihydroperoxyhexadeca-9-enoate | Colletotrichum gloeosporioides | (9Z)-5,8-dihydroxyhexadeca-9-enoic acid | - |
? | |
Subunits
| EC Number | Subunits | Comment | 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 |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.13.11.60 | 8,11-hydroperoxide isomerase | - |
Colletotrichum gloeosporioides |
| 5.4.4.6 | 7,8-diol synthase | - |
Colletotrichum gloeosporioides |
| 5.4.4.6 | 7,8-LDS | - |
Colletotrichum gloeosporioides |
| 5.4.4.6 | 7,8-linoleate diol synthase | - |
Colletotrichum gloeosporioides |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.13.11.60 | 20 | - |
maximum of 8-diogenase reaction | Colletotrichum gloeosporioides |
| 1.13.11.60 | 20 | - |
N-terminal domain activity | Colletotrichum gloeosporioides |
| 1.13.11.60 | 40 | - |
maximum of whole enzyme | Colletotrichum gloeosporioides |
| 1.13.11.60 | 40 | - |
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] | Comment | 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 | Comment | 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-linolenic acid | N-terminal 8-dioxygenase domain, 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 | 17.7 | - |
eicosadienoic 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 | 18.2 | - |
arachidonic 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 | 21.2 | - |
palmitoleate | N-terminal 8-dioxygenase domain, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 21.2 | - |
palmitoleate | recombinant enzyme, whole enzyme 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 | 22 | - |
eicosenoate | recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 24.7 | - |
oleate | N-terminal 8-dioxygenase domain, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 1.13.11.60 | 24.7 | - |
oleate | 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 | - |
linoleate | N-terminal 8-dioxygenase domain, 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 | |
| 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 | Comment | 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 | Colletotrichum gloeosporioides |
| 1.13.11.60 | 6.5 | - |
whole enzyme activity | Colletotrichum gloeosporioides |
| 1.13.11.60 | 7 | - |
maximum of 8-diogenase reaction | Colletotrichum gloeosporioides |
| 1.13.11.60 | 7 | - |
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 | Comment | 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 | |
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Release 2023.1 (January 2023)
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