Information on EC 4.1.99.11 - benzylsuccinate synthase

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The expected taxonomic range for this enzyme is: Bacteria, Archaea

EC NUMBER
COMMENTARY hide
4.1.99.11
-
RECOMMENDED NAME
GeneOntology No.
benzylsuccinate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
benzylsuccinate = toluene + fumarate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
addition
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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Microbial metabolism in diverse environments
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Toluene degradation
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toluene degradation to benzoyl-CoA (anaerobic)
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toluene degradation
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SYSTEMATIC NAME
IUBMB Comments
benzylsuccinate fumarate-lyase (toluene-forming)
A glycyl radical enzyme that is inhibited by benzyl alcohol, benzaldehyde, phenylhydrazine and is inactivated by oxygen.
CAS REGISTRY NUMBER
COMMENTARY hide
209264-18-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
related to Desulfosporosinus sp.
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Manually annotated by BRENDA team
eubacteria, part of a methanogenic consortium
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-
Manually annotated by BRENDA team
strain OX39
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Manually annotated by BRENDA team
strain OX39
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
gene bssA
UniProt
Manually annotated by BRENDA team
gene bssA
UniProt
Manually annotated by BRENDA team
strain TMJ1
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-
Manually annotated by BRENDA team
strain TMJ1
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-
Manually annotated by BRENDA team
strain G5G6
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Manually annotated by BRENDA team
strain G5G6
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-
Manually annotated by BRENDA team
archae, part of a methanogenic consortium
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Manually annotated by BRENDA team
archae, part of a methanogenic consortium
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Manually annotated by BRENDA team
no activity in Dechloromonas aromatica RCB
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-
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Manually annotated by BRENDA team
strain T1, enzyme encoded as part of the tutE tutFDGH gene cluster
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-
Manually annotated by BRENDA team
BssA; gene bssA
UniProt
Manually annotated by BRENDA team
BssA; gene bssA
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
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a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type in sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-fluorotoluene + fumarate
(2-fluorobenzyl)succinate
show the reaction diagram
-
-
-
?
3-fluorotoluene + fumarate
(3-fluorobenzyl)succinate
show the reaction diagram
-
-
-
?
4-fluorotoluene + fumarate
(4-fluorobenzyl)succinate
show the reaction diagram
-
-
-
?
benzaldehyde + fumarate
benzoylsuccinate
show the reaction diagram
-
-
-
?
benzylsuccinate
toluene + fumarate
show the reaction diagram
m-cresol + fumarate
(3-hydroxybenzyl)succinic acid
show the reaction diagram
m-xylene + fumarate
(3-methylbenzyl)succinic acid
show the reaction diagram
m-xylene-d6 + fumarate
(3-methylbenzyl)succinate-d6
show the reaction diagram
-
-
-
?
o-cresol + fumarate
(2-hydroxybenzyl)succinic acid
show the reaction diagram
o-xylene + fumarate
(2-methylbenzyl)succinic acid
show the reaction diagram
o-xylene-d10 + fumarate
(2-methylbenzyl)succinate-d10
show the reaction diagram
-
-
-
?
p-cresol + fumarate
(4-hydroxybenzyl)succinic acid
show the reaction diagram
p-xylene + fumarate
(4-methylbenzyl)succinic acid
show the reaction diagram
p-xylene-d10 + fumarate
(4-methylbenzyl)succinate-d10
show the reaction diagram
-
-
-
?
toluene + fumarate
benzylsuccinate
show the reaction diagram
toluene + maleate
benzylsuccinate
show the reaction diagram
-
-
-
?
[d3-methyl]toluene + fumarate
?
show the reaction diagram
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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
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-
?
[d3-methyl]toluene + maleate
?
show the reaction diagram
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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
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-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
benzylsuccinate
toluene + fumarate
show the reaction diagram
toluene + fumarate
benzylsuccinate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin
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indicated by fluorescence spectroscopy, chemical nature unknown
iron-sulfur centre
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
[4Fe-4S] center
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[4 Fe-4S] clusters in benzylsuccinate synthase are identified via EPR and Mossbauer spectroscopy
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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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
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.11 - 0.35
Toluene
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0024
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cell extract, 4°C
0.003
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0.004
cell extract, pH 7.5, 30°C
0.0074
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cell extract, 30°C
0.015
cell extract, pH 7.5, 30°C
0.017
cell extract, pH 7.5, 30°C
0.0195
cell extract, pH 7.5, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6500
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alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE
7000
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gamma subunit, determined by SDS-PAGE
10000
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alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE
12000
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alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE
14000
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beta subunit, determined by SDS-PAGE
14500
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alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE
90000
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alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE
94000
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alpha, alpha, beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE
97000
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alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE
98000
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98000, SDS-PAGE of the alpha subunit
100000
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alpha subunit, determined by SDS-PAGE
220000
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gel filtration
230000
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determined by gel filtration, consistent with an alpha2beta2gamma2 subunit structure
260000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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98000, SDS-PAGE of the alpha subunit
heterotrimer
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alpha, beta and gamma subunit
hexamer
tetramer
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
extremely oxygen sensitive
rapidly inactivated by oxygen with a half-life of 20-30 s upon exposure to air
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210663
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose, hydroxyapatite, anion exchange chromatography, enzyme severely inactivated during purification
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hydroxyapatite, gel filtration, 95% loss of specific activity after gel filtration
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loss of activity during purification due to high oxygen sensitivity
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using a Ni-NTA-agarose column
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using a Strep-Tactin Superflow column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli as a Strep-tagged fusion protein
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expression in Escherichia coli BL21DE3 cells, the vectors pET-28b, pACYC-Duet, pET-Duet and pRSF-Duet are used
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gene bssA, phylogenetic analysis
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C29S
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tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
C9Y/A10S
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tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
C29S
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tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
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C9Y/A10S
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tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
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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
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site-directed mutagenesis, the mutation affects binding of toluene
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L390A
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site-directed mutagenesis, the mutation affects binding of toluene
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L491A
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site-directed mutagenesis, the mutation affects binding of toluene
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V708A
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site-directed mutagenesis, the mutation affects binding of toluene
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Y829A
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site-directed mutagenesis, the mutation affects binding of toluene
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the alpha subunit is purified and refolded from inclusion bodies
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
environmental protection
additional information