Information on EC 1.1.3.B5 - eugenol oxidase

Substrates: enzyme also oxidizes vanillyl alcohol and vanillylamine, reaction of EC 1.1.3.38. Poor substrates: vanillylamine, 4-ethylguaiacol
Products: -

additional information

Substrates: EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic 4-(1-hydroxyethyl)-2-methoxyphenol. Very poor substrates: 4-(1,3-dithiolan-2-yl)phenol, capsaicin, (4-hydroxyphenyl)acetonitrile, 3-(4-hydroxyphenyl)propanoic acid, 2-methoxy-4-methylphenol, and 4-(hept-1-enyl)-2-methoxyphenol
Products: -

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COFACTOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

FAD

eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. FAD is covalently linked to residue H390. Protein shows absorption maxima at 365 nm and 441 nm, and shoulders at 313 nm, 394 nm, and 461 nm

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KM VALUE [mM]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.00046 - 0.00077

2,6-dimethoxy-4-allylphenol

2 entries

0.0023

4-(Methoxymethyl)phenol

pH 7.5, 25°C

0.0021

4-ethylguaiacol

pH 7.5, 25°C

0.023

5-indanol

pH 7.5, 25°C

0.001

eugenol

pH 7.5, 25°C

0.047

Vanillyl alcohol

His-tagged recombinant enzyme, pH 7.5, 25°C

0.00046

2,6-dimethoxy-4-allylphenol

mutant I427A, pH not specified in the publication, temperature not specified in the publication

0.00077

2,6-dimethoxy-4-allylphenol

wild-type, pH not specified in the publication, temperature not specified in the publication

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TURNOVER NUMBER [1/s]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.37 - 0.72

2,6-dimethoxy-4-allylphenol

2 entries

0.004

4-(Methoxymethyl)phenol

pH 7.5, 25°C

1.7

4-cyclopentylphenol

His-tagged recombinant enzyme, pH 7.5, 25°C

0.026

4-ethylguaiacol

pH 7.5, 25°C

2.4

5-indanol

pH 7.5, 25°C

3.1

eugenol

pH 7.5, 25°C

Vanillyl alcohol

His-tagged recombinant enzyme, pH 7.5, 25°C

0.37

2,6-dimethoxy-4-allylphenol

wild-type, pH not specified in the publication, temperature not specified in the publication

0.72

2,6-dimethoxy-4-allylphenol

mutant I427A, pH not specified in the publication, temperature not specified in the publication

show all columns Go to kcat/KM Value Search

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kcat/KM VALUE [1/mMs^-1]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

4-(Methoxymethyl)phenol

pH 7.5, 25°C

4-ethylguaiacol

pH 7.5, 25°C

100

5-indanol

pH 7.5, 25°C

3100

eugenol

pH 7.5, 25°C

170

Vanillyl alcohol

His-tagged recombinant enzyme, pH 7.5, 25°C

show all columns Go to pH Range Search

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pH RANGE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

9 - 10

more than 90% of maximum activity

show all columns Go to Organism Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks include all textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks + FRENDAco-occurrence of enzyme names and organism names (less precise)

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ORGANISM

COMMENTARY

LITERATURE

UNIPROT

SEQUENCE DB

SOURCE

740440, 762539, 762906, 762907, 763010, 763480

UniProt

brenda

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Highest Expressing Human Cell Lines

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Gene Links

Go to AA Sequence and Transmembrane Helices SearchGo to Localization Prediction

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UNIPROT

ENTRY NAME

ORGANISM

NO. OF AA

NO. OF TRANSM. HELICES

MOLECULAR WEIGHT[Da]

SOURCE

SEQUENCE

LOCALIZATION PREDICTION

The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).

Q0SBK1 pBLAST

Q0SBK1_RHOJR

Rhodococcus jostii (strain RHA1)

526

58681

TrEMBL

Show Sequence

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MOLECULAR WEIGHT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

111000

gel filtration

114000

gel filtration, wild-type

120000

gel filtration, mutant carying a loop of vanillin oxidase

show all columns Go to Subunit Search

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SUBUNIT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

dimer

2 entries

dimer

2 * 58000, SDS-PAGE, 2 * 58681, calculated from sequence

dimer

2 * 60000, wild-type, SDS-PAGE

Go to Crystallization (commentary) Search

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CRYSTALLIZATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

structures in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate. The catalytic center is a remarkable solvent-inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum

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PROTEIN VARIANTS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

G392F

mutant does not display measurable activity

I427A

mutant is significantly more efficient with 2,6-dimethoxy-4-allylphenol than wild-type

I427T

mutant does not display measurable activity

L381W

mutant does not display measurable activity

L438C

variant displays lowered activity towards all substrates as compared to wild-type

M282L

substrate specificity profile is similar to wild-type

Q425T

display similar activity to the wild-type enzyme with vanillyl alcohol, but no measurable activity towards any other substrate

V436I

variant displays lowered activity towards vanillyl alcohol and eugenol

additional information

exchange of a loop at the dimer-dimer interface in octameric vanillin oxidase that is not present in dimeric EUGO. A vanillin oxidase variant where the loop was deleted, loopless VAO, exclusively forms dimers. Introduction of the loop into EUGO is not sufficient to induce its octamerization. Neither variant displays major changes in its catalytic properties as compared to the wild-type enzyme

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TEMPERATURE STABILITY

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

0.0034 mM enzyme in in 20 mm Tris/HCl, pH 7.5 for 90 min, no inactivation

half-life 30 min, in presence of 3fold excess of FAD half-life 45 min

melting temperature, pH 5-8. The presence of 10% (v/v) DMSO or ethyl acetate does not significantly affect the enzyme's thermostability

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ORGANIC SOLVENT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

diisopropyl ether

increases selectivtiy and conversion rate of the reaction with 4-(1-hydroxyethyl)-2-methoxyphenol

DMSO

10% (v/v), increase in the enzymatic activity

Ethyl acetate

increase in enzymatic activity

Go to Purification (commentary) Search

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PURIFICATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

recombinant enzyme

Go to Cloned (commentary) Search

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CLONED (Commentary)

ORGANISM

UNIPROT

LITERATURE

expression in Escherichia coli

show all columns Go to Application Search

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APPLICATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

analysis

development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method

synthesis

synthesis

fusion of eugenol oxidase and 5-hydroxymethylfurfural oxidase to be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers

762907

synthesis

synthesis of syringaresinol in a one-pot conversion containing eugenol oxidase (EUGO) and horseradish peroxidase (HRP) using 2,6-dimethoxy-4-allylphenol as a substrate. The hydrogen peroxide generated from the reaction of EUGO with the substrate is utilized by the HRP to convert the formed sinapyl alcohol into syringaresinol. Mutant I427A together with HRP are capable of efficiently producing syringaresinol as a major product. After optimization and upscaling to a semipreparative scale (1 gr), syringaresinol is obtained in 81% yield

top print hide References Search

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REF.

AUTHORS

TITLE

JOURNAL

VOL.

PAGES

YEAR

ORGANISM (UNIPROT)

PUBMED ID

SOURCE

Ewing, T.A.; Gygli, G.; van Berkel, W.J.

A single loop is essential for the octamerization of vanillyl alcohol oxidase

FEBS J.

283

2546-2559

2016

Rhodococcus jostii (Q0SBK1)

27214042

brenda

Habib, M.; Trajkovic, M.; Fraaije, M.W.

The biocatalytic synthesis of syringaresinol from 2,6-dimethoxy-4-allylphenol in one-pot using a tailored oxidase/peroxidase system

ACS Catal.

5549-5552

2018

Rhodococcus jostii (Q0SBK1)

29888098

brenda

Nguyen, Q.-T.; de Gonzalo, G.; Binda, C.; Rioz-Martinez, A.; Mattevi, A.; Fraaije, M.W.

Biocatalytic properties and structural analysis of eugenol oxidase from Rhodococcus jostii RHA1 A versatile oxidative biocatalyst

Chembiochem

1359-1366

2016

Rhodococcus jostii (Q0SBK1)

27123962

brenda

762907

Colpa, D.I.; Loncar, N.; Schmidt, M.; Fraaije, M.W.

Creating oxidase-peroxidase fusion enzymes as a toolbox for cascade reactions

Chembiochem

2226-2230

2017

Rhodococcus jostii (Q0SBK1)

28885767

brenda

Jin, J.; Mazon, H.; van den Heuvel, R.H.H.; Janssen, D.B.; Fraaije, M.W.

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1

FEBS J.

274

2311-2321

2007

Rhodococcus jostii (Q0SBK1)

17419730

brenda