Information on EC 4.1.99.11 - benzylsuccinate synthase

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2-fluorotoluene + fumarate

(2-fluorobenzyl)succinate

3-fluorotoluene + fumarate

(3-fluorobenzyl)succinate

4-fluorotoluene + fumarate

(4-fluorobenzyl)succinate

benzaldehyde + fumarate

benzoylsuccinate

benzylsuccinate

toluene + fumarate

12 entries

m-cresol + fumarate

(3-hydroxybenzyl)succinic acid

m-xylene + fumarate

(3-methylbenzyl)succinic acid

m-xylene-d6 + fumarate

(3-methylbenzyl)succinate-d6

o-cresol + fumarate

(2-hydroxybenzyl)succinic acid

o-xylene + fumarate

(2-methylbenzyl)succinic acid

o-xylene-d10 + fumarate

(2-methylbenzyl)succinate-d10

p-cresol + fumarate

(4-hydroxybenzyl)succinic acid

p-xylene + fumarate

(4-methylbenzyl)succinic acid

p-xylene-d10 + fumarate

(4-methylbenzyl)succinate-d10

toluene + fumarate

benzylsuccinate

32 entries

toluene + maleate

benzylsuccinate

[d3-methyl]toluene + fumarate

deuterium is transferred from toluene to the C-3 pro-(S) position of benzylsuccinate, implying that the addition of toluene to the double bond of fumarate is syn

665307

[d3-methyl]toluene + maleate

addition of toluene occurs in anti fashion. Formation of the C-3 radical of the benzylsuccinate as an intermediate, in which rotation about the C-2-C-3 bond can occur to relieve the sterically unfavorable cis conformation

665307

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benzylsuccinate

toluene + fumarate

10 entries

toluene + fumarate

benzylsuccinate

27 entries

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flavin

indicated by fluorescence spectroscopy, chemical nature unknown

iron-sulfur centre

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[4Fe-4S] center

Escherichia coli

[4 Fe-4S] clusters in benzylsuccinate synthase are identified via EPR and Mossbauer spectroscopy

715671

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additional information

tutE gene product (benzylsuccinate synthase activating enzyme) required for activity, changes in the tutE encoded protein (C72A and C79A) are able to prevent the action of benzylsuccinate synthase; tutH gene product required for activity, changes in the tutH encoded protein (G52A/K53R/S54A) are able to prevent the action of benzylsuccinate synthase, tutH gene product possibly involved in assembly of the enzyme subunits

677932

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0.11 - 0.35

Toluene

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0.0002

0.0024

cell extract, 4°C

0.003

0.004

Desulfosarcina cetonica

A2TBP7;

cell extract, pH 7.5, 30°C

0.0074

cell extract, 30°C

Geobacter metallireducens

0.015

Q39VF1;

cell extract, pH 7.5, 30°C

0.016

3 entries

0.017

Q8VPT7;

cell extract, pH 7.5, 30°C

0.0195

O87943;

cell extract, pH 7.5, 30°C

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7.5

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culture condition:toluene-grown cell

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665307

additional information

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Thauera aromatica;

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6500

alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE

7000

gamma subunit, determined by SDS-PAGE

10000

alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE

12000

alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE

14000

beta subunit, determined by SDS-PAGE

14500

alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE

90000

alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE

94000

alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE

97000

alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE

98000

98000, SDS-PAGE of the alpha subunit

100000

alpha subunit, determined by SDS-PAGE

220000

gel filtration

230000

determined by gel filtration, consistent with an alpha2beta2gamma2 subunit structure

260000

gel filtration

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98000, SDS-PAGE of the alpha subunit

heterotrimer

alpha, beta and gamma subunit

hexamer

tetramer

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extremely oxygen sensitive

7 entries

rapidly inactivated by oxygen with a half-life of 20-30 s upon exposure to air

210663

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DEAE-Sepharose, hydroxyapatite, anion exchange chromatography, enzyme severely inactivated during purification

hydroxyapatite, gel filtration, 95% loss of specific activity after gel filtration

loss of activity during purification due to high oxygen sensitivity

using a Ni-NTA-agarose column

using a Strep-Tactin Superflow column

Escherichia coli

715671

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expressed in Escherichia coli

expressed in Escherichia coli as a Strep-tagged fusion protein

Escherichia coli

715671

expression in Escherichia coli BL21DE3 cells, the vectors pET-28b, pACYC-Duet, pET-Duet and pRSF-Duet are used

gene bssA, phylogenetic analysis

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C29S

tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source

677932

C9Y/A10S

tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source

677932

C29S

Thauera aromatica T1

tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source

C9Y/A10S

Thauera aromatica T1

tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source

E509A

site-directed mutagenesis, the mutation affects binding of fumarate

F384A

site-directed mutagenesis, the mutation affects binding of toluene

L390A

site-directed mutagenesis, the mutation affects binding of toluene

L491A

site-directed mutagenesis, the mutation affects binding of toluene

Q706A

site-directed mutagenesis, the mutation affects binding of fumarate

S827A

site-directed mutagenesis, the mutation affects binding of fumarate

V708A

site-directed mutagenesis, the mutation affects binding of toluene

Y829A

site-directed mutagenesis, the mutation affects binding of toluene

F384A

site-directed mutagenesis, the mutation affects binding of toluene

L390A

site-directed mutagenesis, the mutation affects binding of toluene

L491A

site-directed mutagenesis, the mutation affects binding of toluene

V708A

site-directed mutagenesis, the mutation affects binding of toluene

Y829A

site-directed mutagenesis, the mutation affects binding of toluene

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the alpha subunit is purified and refolded from inclusion bodies