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

expression in Escherichia coli; phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566

Colletotrichum gloeosporioides

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

C1038S

site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE

Colletotrichum gloeosporioides

H1036A

site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE

Colletotrichum gloeosporioides

H1036A/C1038S

site-directed mutagenesis, the double mutant converts linoleic acid to only 8-HPODE

Colletotrichum gloeosporioides

comparison of substrate specificities of wild-type an d mutant enzymes, overview

Colletotrichum gloeosporioides

N895D

site-directed mutagenesis, the mutant produces a reduced amount of 8-HPODE, compared to wild-type, without the accumulation of DiHODE

Colletotrichum gloeosporioides

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

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

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

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

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

0.0193

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0193

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.022

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

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

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

0.0319

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0319

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0324

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0324

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0333

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0358

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0358

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0368

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0369

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0371

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.048

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

510000

gel filtration; recombinant enzyme, gel filtration

Colletotrichum gloeosporioides

linoleate + O2

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

?

DMSO

maximum activity in presence of 2.5% DMSO

Colletotrichum gloeosporioides

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

Colletotrichum gloeosporioides KACC 40961

A0A0S2SWE4

i.e. Colletotrichum gloeosporioides

recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography

Colletotrichum gloeosporioides

mycelium

Colletotrichum gloeosporioides

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

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

1.17

purified recombinant enzyme, N-terminal domain activity, substrate arachidate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.18

purified recombinant enzyme, N-terminal domain activity, substrate dihomo-gamma-linolenate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.2

purified recombinant enzyme, N-terminal domain activity, substrate eicosadienoate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.39

purified recombinant enzyme, N-terminal domain activity, substrate eicosenoate, pH 6.5, 40°C

Colletotrichum gloeosporioides

alpha-linolenate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxy-9,12,15-octadecatrienoate

?

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

?

arachidonate + O2

741712

Colletotrichum gloeosporioides

(5Z,8Z,11Z,14Z)-8-hydroxyeicosa-5,8,11,14-tetraenoic acid

?

arachidonic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyarachidonic acid

?

dihomo-gamma-linolenate + O2

741712

Colletotrichum gloeosporioides

(8Z,11Z,12Z)-8-hydroxyeicosa-8,11,12-trienoic acid

?

dihomo-gamma-linolenic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxydihomo-gamma-linolenic acid

?

eicosadienoate + O2

741712

Colletotrichum gloeosporioides

(11Z,14Z)-8-hydroxyeicosa-11,14-dienoic acid

?

eicosadienoic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyeicosadienoic acid

?

eicosenoate + O2

741712

Colletotrichum gloeosporioides

(11Z)-8-hydroxyeicosa-11-enoic acid

?

eicosenoic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyeicosenoic acid

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(7S,8S)-7,8-dihydroxy-(9Z,12Z)-octadecadienoic acid

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(R)-8-hydroperoxy-(9Z,12Z)-octadecadienoic acid

?

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

?

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

?

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

?

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

?

oleate + O2

741712

Colletotrichum gloeosporioides

(9Z)-8-hydroperoxyoctadec-9-enoate

?

oleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(9Z)-8-hydroperoxyoctadec-9-enoate

?

oleate + O2

via (9Z)-8-hydroperoxyoctadeca-9-enoic acid

741712

Colletotrichum gloeosporioides

(9Z)-7,8-dihydroxyoctadeca-9-enoic acid

?

palmitoleate + O2

741712

Colletotrichum gloeosporioides

(9Z)-8-hydroperoxyhexadec-9-enoate

?

palmitoleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(9Z)-8-hydroperoxyhexadec-9-enoate

?

palmitoleate + O2

via (9Z)-dihydroperoxyhexadeca-9-enoate

741712

Colletotrichum gloeosporioides

(9Z)-5,8-dihydroxyhexadeca-9-enoic acid

?

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

trimer

3 * 127000, SDS-PAGE, 3 * 127413, calculated

Colletotrichum gloeosporioides

20

maximum of 8-diogenase reaction; N-terminal domain activity

Colletotrichum gloeosporioides

40

maximum of whole enzyme; whole enzyme activity

Colletotrichum gloeosporioides

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

4.86

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

5.62

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

6.58

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

10.5

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.2

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.2

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.7

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.7

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

18.2

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

18.2

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

22

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

22

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

25.1

alpha-linolenate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

25.2

alpha-linolenate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

35

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

35

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

maximum of bifunctional enzyme; whole enzyme activity

Colletotrichum gloeosporioides

7

maximum of 8-diogenase reaction; N-terminal domain activity

Colletotrichum gloeosporioides

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

expression in Escherichia coli; phylogenetic tree, recombinant expression of wild-type and mutant His-tagged enzymes in Escherichia coli strain ER2566

Colletotrichum gloeosporioides

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

C1038S

site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE

Colletotrichum gloeosporioides

H1036A

site-directed mutagenesis, the mutant produces a low amount of 7,8-DiHODE with the accumulation of 8-HPODE

Colletotrichum gloeosporioides

H1036A/C1038S

site-directed mutagenesis, the double mutant converts linoleic acid to only 8-HPODE

Colletotrichum gloeosporioides

comparison of substrate specificities of wild-type an d mutant enzymes, overview

Colletotrichum gloeosporioides

N895D

site-directed mutagenesis, the mutant produces a reduced amount of 8-HPODE, compared to wild-type, without the accumulation of DiHODE

Colletotrichum gloeosporioides

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

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

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

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

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

0.0193

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0193

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.022

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

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

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

0.0319

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0319

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0324

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0324

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0333

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0358

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0358

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0368

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0369

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.0371

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

0.048

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

510000

gel filtration; recombinant enzyme, gel filtration

Colletotrichum gloeosporioides

linoleate + O2

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

?

DMSO

maximum activity in presence of 2.5% DMSO

Colletotrichum gloeosporioides

recombinant His-tagged wild-type and mutant enzymes 8fold from Escherichia coli strain ER2566 by nickel affinity chromatography

Colletotrichum gloeosporioides

mycelium

Colletotrichum gloeosporioides

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

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

1.17

purified recombinant enzyme, N-terminal domain activity, substrate arachidate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.18

purified recombinant enzyme, N-terminal domain activity, substrate dihomo-gamma-linolenate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.2

purified recombinant enzyme, N-terminal domain activity, substrate eicosadienoate, pH 6.5, 40°C

Colletotrichum gloeosporioides

1.39

purified recombinant enzyme, N-terminal domain activity, substrate eicosenoate, pH 6.5, 40°C

Colletotrichum gloeosporioides

alpha-linolenate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxy-9,12,15-octadecatrienoate

?

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

?

arachidonate + O2

741712

Colletotrichum gloeosporioides

(5Z,8Z,11Z,14Z)-8-hydroxyeicosa-5,8,11,14-tetraenoic acid

?

arachidonic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyarachidonic acid

?

dihomo-gamma-linolenate + O2

741712

Colletotrichum gloeosporioides

(8Z,11Z,12Z)-8-hydroxyeicosa-8,11,12-trienoic acid

?

dihomo-gamma-linolenic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxydihomo-gamma-linolenic acid

?

eicosadienoate + O2

741712

Colletotrichum gloeosporioides

(11Z,14Z)-8-hydroxyeicosa-11,14-dienoic acid

?

eicosadienoic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyeicosadienoic acid

?

eicosenoate + O2

741712

Colletotrichum gloeosporioides

(11Z)-8-hydroxyeicosa-11-enoic acid

?

eicosenoic acid + O2

741712

Colletotrichum gloeosporioides

8-hydroxyeicosenoic acid

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(8R,9Z,12Z)-8-hydroperoxy-9,12-octadecadienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(7S,8S)-7,8-dihydroxy-(9Z,12Z)-octadecadienoic acid

?

linoleate + O2

741712

Colletotrichum gloeosporioides

(R)-8-hydroperoxy-(9Z,12Z)-octadecadienoic acid

?

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

?

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

?

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

?

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

?

oleate + O2

741712

Colletotrichum gloeosporioides

(9Z)-8-hydroperoxyoctadec-9-enoate

?

oleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(9Z)-8-hydroperoxyoctadec-9-enoate

?

oleate + O2

via (9Z)-8-hydroperoxyoctadeca-9-enoic acid

741712

Colletotrichum gloeosporioides

(9Z)-7,8-dihydroxyoctadeca-9-enoic acid

?

palmitoleate + O2

741712

Colletotrichum gloeosporioides

(9Z)-8-hydroperoxyhexadec-9-enoate

?

palmitoleate + O2

741712

Colletotrichum gloeosporioides KACC 40961

(9Z)-8-hydroperoxyhexadec-9-enoate

?

palmitoleate + O2

via (9Z)-dihydroperoxyhexadeca-9-enoate

741712

Colletotrichum gloeosporioides

(9Z)-5,8-dihydroxyhexadeca-9-enoic acid

?

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

trimer

3 * 127000, SDS-PAGE, 3 * 127413, calculated

Colletotrichum gloeosporioides

20

maximum of 8-diogenase reaction; N-terminal domain activity

Colletotrichum gloeosporioides

40

maximum of whole enzyme; whole enzyme activity

Colletotrichum gloeosporioides

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

4.86

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

5.62

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

6.58

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

10.5

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.2

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.2

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.7

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

17.7

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

18.2

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

18.2

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

22

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

22

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

25.1

alpha-linolenate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

25.2

alpha-linolenate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

35

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

35

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

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

maximum of bifunctional enzyme; whole enzyme activity

Colletotrichum gloeosporioides

7

maximum of 8-diogenase reaction; N-terminal domain activity

Colletotrichum gloeosporioides

100

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

170

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

340

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

370

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

466.1

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

470

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

508.4

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

510

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

554.9

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

560

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

679

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

680

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

690

palmitoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

695.1

palmitoleate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

840

oleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

843

oleate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

950

alpha-linolenate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

954.54

alpha-linolenate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

1813.5

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

1820

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

100

palmitoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

170

oleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

340

alpha-linolenate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

370

linoleate

bifunctional enzyme, pH 6.5, 40°C

Colletotrichum gloeosporioides

466.1

dihomo-gamma-linolenate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

470

dihomo-gamma-linolenic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

508.4

arachidonate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

510

arachidonic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

554.9

eicosadienoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

560

eicosadienoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

679

eicosenoate

recombinant enzyme, N-terminal domain activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

680

eicosenoic acid

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

690

palmitoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

695.1

palmitoleate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

840

oleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

843

oleate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

950

alpha-linolenate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

954.54

alpha-linolenate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

1813.5

(8R,9Z,12Z)-8-hydroperoxyoctadeca-9,12-dienoate

recombinant enzyme, whole enzyme activity, pH 6.5, 40°C

Colletotrichum gloeosporioides

1820

linoleate

N-terminal 8-dioxygenase domain, pH 6.5, 40°C

Colletotrichum gloeosporioides

742730

Seo

Production of 7,8-dihydroxy u ...

J. Agric. Food Chem.

743200

Sooman

Discovery of a novel linoleat ...

Lipids

724132

Hoffmann

7,8- and 5,8-linoleate diol sy ...

Arch. Biochem. Biophys.

711024

Hoffmann

Expression of 5,8-LDS of Asper ...

Arch. Biochem. Biophys.

714317

Jernerén

Reaction mechanism of 5,8-lino ...

Biochim. Biophys. Acta

704466

Brodhun

Identification of PpoA from As ...

J. Biol. Chem.

704475

Garscha

Leucine/valine residues direct ...

J. Biol. Chem.

702018

Garscha

Pichia expression and mutagene ...

Biochem. Biophys. Res. Commun.

703700

Garscha

Critical amino acids for the 8 ...

FEBS Lett.

285246

Hörnsten

Cloning of linoleate diol synt ...

J. Biol. Chem.

285251

Oliw

Catalytic properties of linole ...

Adv. Exp. Med. Biol.

285244

Su

A protein radical and ferryl i ...

J. Biol. Chem.

285243

Su

Purification and characterizat ...

J. Biol. Chem.

285249

Su

Studies on linoleic acid 8R-di ...

Lipids

285247

Brodowsky

BW A4C and other hydroxamic ac ...

Eur. J. Pharmacol.

285248

Brodowsky

Linoleic acid 8R-dioxygenase a ...

Ann. N. Y. Acad. Sci.

285250

Hamberg

Sequential oxygenation of lino ...

Arch. Biochem. Biophys.

285245

Brodowsky

A linoleic acid (8R)-dioxygena ...

J. Biol. Chem.