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Literature summary for 5.4.4.6 extracted from
- Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate (2016), Appl. Microbiol. Biotechnol., 100, 3087-3099 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566 | Colletotrichum gloeosporioides |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| A891G | site-directed mutagenesis | Colletotrichum gloeosporioides |
| C1038S | site-directed mutagenesis | Colletotrichum gloeosporioides |
| H1036A | site-directed mutagenesis | Colletotrichum gloeosporioides |
| H1036A/C1038S | site-directed mutagenesis | Colletotrichum gloeosporioides |
| additional information | comparison of substrate specificities of wild-type and mutant enzymes, overview | Colletotrichum gloeosporioides |
| N895D | site-directed mutagenesis | Colletotrichum gloeosporioides |
| N895L | site-directed mutagenesis | Colletotrichum gloeosporioides |
| N895Q | site-directed mutagenesis | Colletotrichum gloeosporioides |
| Q898E | site-directed mutagenesis | Colletotrichum gloeosporioides |
| Q898L | site-directed mutagenesis | Colletotrichum gloeosporioides |
| Y380F | site-directed mutagenesis | Colletotrichum gloeosporioides |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0223 | - |
alpha-linolenate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides |  |
| 0.0333 | - |
oleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 0.0371 | - |
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 0.048 | - |
palmitoleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 510000 | - |
recombinant enzyme, gel filtration | Colletotrichum gloeosporioides |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | Colletotrichum gloeosporioides | - |
(7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Colletotrichum gloeosporioides | A0A0S2SWE4 | i.e. Colletotrichum gloeosporioides | - |
| Colletotrichum gloeosporioides KACC 40961 | A0A0S2SWE4 | i.e. Colletotrichum gloeosporioides | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography | Colletotrichum gloeosporioides |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| mycelium | - |
Colletotrichum gloeosporioides | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.39 | - |
purified recombinant enzyme, whole enzyme activity, substrate palmitoleate, pH 6.5, 40°C | Colletotrichum gloeosporioides |
| 0.48 | - |
purified recombinant enzyme, whole enzyme activity, substrate oleate, pH 6.5, 40°C | Colletotrichum gloeosporioides |
| 0.75 | - |
purified recombinant enzyme, whole enzyme activity, substrate alpha-linolenate, pH 6.5, 40°C | Colletotrichum gloeosporioides |
| 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)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | - |
Colletotrichum gloeosporioides | (7S,8S,9Z,12Z)-7,8-dihydroxyoctadeca-9,12-dienoate | - |
r | |
| 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 | - |
? | |
| 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 | ? | - |
? | |
| oleate + O2 | via (9Z)-8-hydroperoxyoctadeca-9-enoic acid | Colletotrichum gloeosporioides | (9Z)-7,8-dihydroxyoctadeca-9-enoic acid | - |
? | |
| palmitoleate + O2 | via (9Z)-8-dihydroperoxyhexadeca-9-enoate | Colletotrichum gloeosporioides | (9Z)-5,8-dihydroxyhexadeca-9-enoic acid | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homotetramer | 4 * 127000, recombinant His-tagged enzyme, SDS-PAGE, 4 * 127413, sequence calculation | Colletotrichum gloeosporioides |
| 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
| Synonyms | Comment | Organism |
|---|---|---|
| 7,8-diol synthase | - |
Colletotrichum gloeosporioides |
| 7,8-LDS | - |
Colletotrichum gloeosporioides |
| 7,8-linoleate diol synthase | - |
Colletotrichum gloeosporioides |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | - |
whole enzyme activity | Colletotrichum gloeosporioides |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 4.86 | - |
palmitoleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 5.62 | - |
oleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 6.58 | - |
alpha-linolenate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 10.5 | - |
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 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 |
| 6.5 | - |
whole enzyme activity | Colletotrichum gloeosporioides |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 8.125 | - |
palmitoleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 14.41 | - |
oleate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 23.45 | - |
(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
| 33.62 | - |
alpha-linolenate | recombinant enzyme, whole enzyme activity, pH 6.5, 40°C | Colletotrichum gloeosporioides | |
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