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Information on EC 4.1.99.11 - benzylsuccinate synthase
for references in articles please use BRENDA:EC4.1.99.11
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EC Tree 4 Lyases
4.1 Carbon-carbon lyases
4.1.99 Other carbon-carbon lyases
4.1.99.11 benzylsuccinate synthase
IUBMB Comments
A glycyl radical enzyme that is inhibited by benzyl alcohol, benzaldehyde, phenylhydrazine and is inactivated by oxygen.
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
- 4.1.99.11
-
denitrifying
-
glycyl
- thauera
- aromatica
-
toluene-degrading
-
sulfate-reducing
-
aquifer
- benzoyl-coa
- azoarcus
- ethylbenzene
- geobacter
-
r-benzylsuccinate
-
hydrocarbon-degrading
-
plume
- m-xylene
- formate-lyase
- environmental protection
-
iron-reducing
- 2-methylnaphthalene
-
hydrocarbon-contaminated
-
feiii-reducing
-
toluene-induced
- desulfobulbaceae
-
nitrate-reducing
-
alkylbenzenes
-
toluene-grown
-
compound-specific
- magnetospirillum
-
alkylsuccinate
showSynonyms
benzylsuccinate synthase, toluene-activating enzyme, (r)-benzylsuccinate synthase, benzylsuccinate synthase a, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(R)-benzylsuccinate synthase
benzylsuccinate synthase
benzylsuccinate synthase A
BSS
BSSA
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
benzylsuccinate = toluene + fumarate
stereospecific radical addition of toluene to fumarate, enzyme carries a stable organic free radical, most probably located on glycine residue 828
-
benzylsuccinate = toluene + fumarate
substrate binding stabilizing the active site and reaction mechanism via glycyl radical, detailed overview. Syn addition of toluene to fumaric acid and facilitation of a mechanism that retains the hydrogen abstracted from the methyl group of toluene within the succinyl moiety. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition
Q8L1A3
benzylsuccinate = toluene + fumarate
reaction mechanism of enzymatic radical C-C coupling by benzylsuccinate synthase, molecular dynamics (MD) simulations and quantum mechanics (QM) modeling, detailed overview. The enzyme is a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro-(R)-orientation and reverse preference of benzyl radical attack on fumarate in pro-(S)-pathway which results with prohibitively high energy barrier of the radical quenching
benzylsuccinate = toluene + fumarate
benzylsuccinate synthase (BSS) catalyzes the formation of a C-C bond between toluene and fumarate by a radical mechanism. BSS binds substrates in a buried active site and uses conformational changes to gate access. The C-C bond-forming reaction performed by BSS requires an oxygen-sensitive radical cofactor. BSS contains a backbone glycyl radical in its activated form. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation, the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSbeta, a small protein subunit that forms a tight complex with BSSalpha, the catalytic subunit. Substrates can enter the active site through a channel, which can be blocked by subunit beta, BSSbeta plays a role in gating active site accessibility
benzylsuccinate = toluene + fumarate
DFT model of the reaction mechanism of BSS, mechanistic modeling
benzylsuccinate = toluene + fumarate
DFT model of the reaction mechanism of BSS, mechanistic modeling
I6LHT8
benzylsuccinate = toluene + fumarate
DFT model of the reaction mechanism of BSS, mechanistic modeling
-
benzylsuccinate = toluene + fumarate
benzylsuccinate synthase (BSS) catalyzes the formation of a C-C bond between toluene and fumarate by a radical mechanism, overview. An enzyme-bound glycyl radical cofactor transiently forms a catalytically essential enzyme-bound thiyl (Cys) radical
benzylsuccinate = toluene + fumarate
stereospecific radical addition of toluene to fumarate, enzyme carries a stable organic free radical, most probably located on glycine residue 828
-
-
benzylsuccinate = toluene + fumarate
DFT model of the reaction mechanism of BSS, mechanistic modeling
-
-
benzylsuccinate = toluene + fumarate
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
addition
addition
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
MetaCyc
toluene degradation to benzoyl-CoA (anaerobic)
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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.
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CAS REGISTRY NUMBER
COMMENTARY
209264-18-6
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-fluorotoluene + fumarate
(2-fluorobenzyl)succinate
-
Substrates: -
Products: -
Products: -
?
3-fluorotoluene + fumarate
(3-fluorobenzyl)succinate
-
Substrates: -
Products: -
Products: -
?
4-fluorotoluene + fumarate
(4-fluorobenzyl)succinate
-
Substrates: -
Products: -
Products: -
?
m-xylene-d6 + fumarate
(3-methylbenzyl)succinate-d6
-
Substrates: -
Products: -
Products: -
?
o-xylene-d10 + fumarate
(2-methylbenzyl)succinate-d10
-
Substrates: -
Products: -
Products: -
?
p-xylene-d10 + fumarate
(4-methylbenzyl)succinate-d10
-
Substrates: -
Products: -
Products: -
?
[d3-methyl]toluene + fumarate
?
-
Substrates: 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
Products: -
Products: -
?
[d3-methyl]toluene + maleate
?
-
Substrates: 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
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Geobacter metallireducens GS-15 / ATCC 53774 / DSM 7210
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Q8L1A3
Substrates: enzyme-substrate interactions at the active site by molecular dynamics simulations, overview
Products: involvement of Phe384, Leu390, Leu491, Tyr829, and Val708 in stabilizing toluene binding and Ser827, Glu509, and Gln706 in stabilizing fumaric acid binding
Products: involvement of Phe384, Leu390, Leu491, Tyr829, and Val708 in stabilizing toluene binding and Ser827, Glu509, and Gln706 in stabilizing fumaric acid binding
?
m-cresol + fumarate
(3-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
m-cresol + fumarate
(3-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
m-xylene + fumarate
(3-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
m-xylene + fumarate
(3-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
m-xylene + fumarate
(3-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-cresol + fumarate
(2-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-cresol + fumarate
(2-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-cresol + fumarate
(2-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-cresol + fumarate
(2-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-xylene + fumarate
(2-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-xylene + fumarate
(2-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
o-xylene + fumarate
(2-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
p-cresol + fumarate
(4-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
p-cresol + fumarate
(4-hydroxybenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
p-xylene + fumarate
(4-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
p-xylene + fumarate
(4-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
p-xylene + fumarate
(4-methylbenzyl)succinic acid
-
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: on incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) is coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia)
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99%
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
ir
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
Substrates: the enzyme generates enantiospecifically (R)-benzylsuccinate from fumarate and toluene
Products: -
Products: -
r
toluene + fumarate
benzylsuccinate
Substrates: benzylsuccinate synthase (BSS) catalyzes the formation of a C-C bond between toluene and fumarate by a radical mechanism. BSS binds substrates in a buried active site and uses conformational changes to gate access. Both substrates can bind to the BSSalphabetagamma complex
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
additional information
?
-
Substrates: docking of the substrates into the relaxed active site and structure of the enzyme-product complex, overview. The active site cavity contains two hydrophobic regions, which are involved in interacting with the benzene ring and the protonated carboxyl group, respectively. On the other hand, a hydrophilic portion of the active site cavity binds the deprotonated carboxylic group by a strong electrostatic interaction towards Arg508 and a range of hydrogen bonds. Kinetic isotope effect of 4.0 for [2H] substituents at the methyl group of toluene
Products: -
Products: -
?
additional information
?
-
-
Substrates: docking of the substrates into the relaxed active site and structure of the enzyme-product complex, overview. The active site cavity contains two hydrophobic regions, which are involved in interacting with the benzene ring and the protonated carboxyl group, respectively. On the other hand, a hydrophilic portion of the active site cavity binds the deprotonated carboxylic group by a strong electrostatic interaction towards Arg508 and a range of hydrogen bonds. Kinetic isotope effect of 4.0 for [2H] substituents at the methyl group of toluene
Products: -
Products: -
?
additional information
?
-
Substrates: substrate-bound structures of benzylsuccinate synthase, fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation, the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSbeta, a small protein subunit that forms a tight complex with BSSalpha, the catalytic subunit. Toluene is activated in anaerobic hydrocarbon degradation, overview. Fumarate binding stabilizes a partially closed conformation in the dimeric BSSalphagamma
Products: -
Products: -
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Substrates: -
Products: -
Products: -
?
benzylsuccinate
toluene + fumarate
Geobacter metallireducens GS-15 / ATCC 53774 / DSM 7210
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: highly stereospecific, enantiomer purity for (R-)-(+)-benzylsuccinate 99%
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
ir
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
Substrates: -
Products: -
Products: -
?
toluene + fumarate
benzylsuccinate
-
Substrates: first step in the anaerobic degradation pathway of toluene
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
the BSS small subunits resemble high potential iron-sulfur proteins
-
glycyl radical
the C-C bond-forming reaction performed by BSS and its relatives requires an oxygen-sensitive radical cofactor, mechanism, overview
glycyl radical
an enzyme-bound glycyl radical cofactor transiently forms a catalytically essential enzyme-bound thiyl (Cys) radical
[4Fe-4S] cluster
one low potential [4Fe4S]-cluster is located in each of the two small subunits
[4Fe-4S] cluster
a [4Fe4S]-cluster is located in the small subunit beta, while there is no bound [4Fe-4S] cluster observable in the electron density of subunit BSSgamma
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
[4Fe-4S] center
-
[4 Fe-4S] clusters in benzylsuccinate synthase are identified via EPR and Mossbauer spectroscopy
Fe2+
one low potential [4Fe4S]-cluster is located in each of the two small subunits
Fe2+
one [4Fe4S]-cluster is located in the small subunit beta
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
-
additional information
-
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
-
additional information
enzyme BSS needs to be posttranslationally activated to the active, radical-containing state by a separate activating enzyme, which is encoded in a common operon with the genes for the BSS subunits and belongs to the family of S-adenosyl-methionine-dependent radical enzymes
-
additional information
-
enzyme BSS needs to be posttranslationally activated to the active, radical-containing state by a separate activating enzyme, which is encoded in a common operon with the genes for the BSS subunits and belongs to the family of S-adenosyl-methionine-dependent radical enzymes
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.016
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
alpha-, beta-, and gamma-subunit
UniProt
brenda
alpha-, beta-, and gamma-subunit
UniProt
brenda
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
metabolism
physiological function
additional information
evolution
enzyme BSS is a member of the glycyl radical enzyme (GRE) family
evolution
enzyme BSS is a member of the glycyl radical enzyme (GRE) family. Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes, phylogenetic analysis, overview
evolution
I6LHT8
enzyme BSS is a member of the glycyl radical enzyme (GRE) family. Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes, phylogenetic analysis, overview
evolution
-
enzyme BSS is a member of the glycyl radical enzyme (GRE) family. Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes, phylogenetic analysis, overview
evolution
enzyme BSS is a member of the glycyl radical enzyme (GRE) family
evolution
-
enzyme BSS is a member of the glycyl radical enzyme (GRE) family. Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes, phylogenetic analysis, overview
-
metabolism
-
BSSA gene encoding the alpha-subunit of Bss from a novel taxon of the Betaproteobacteria catalyzes the first step in anaerobic toluene degradation
metabolism
-
BSSA gene encoding the alpha-subunit of Bss from a novel taxon of the Betaproteobacteria catalyzes the first step in anaerobic toluene degradation
-
physiological function
-
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
physiological function
the enzyme is a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica
physiological function
benzylsuccinate synthase (BSS) catalyzes the first step in anaerobic toluene degradation, the generation of (R)-benzylsuccinate from toluene and fumarate. This unusual reaction is thought to proceed through radical mediated C-C bond formation, with the radical source being an enzyme-bound glycyl radical cofactor that transiently forms a catalytically essential enzyme-bound thiyl (Cys) radical
additional information
Q8L1A3
the catalytic subunit has the putative radical sites located on Cys492 and Gly828, induced-fit product docking approach and substrate-bound molecular dynamics simulations based on the refined active site topology, three-dimensional structure homology modeling, overview
additional information
the C-C bond-forming reaction performed by BSS and its relatives requires an oxygen-sensitive radical cofactor. BSS is a member of the glycyl radical enzyme (GRE) family and contains a backbone glycyl radical in its activated form
additional information
enzyme structure and structure-based mechanistic models, overview. The QM cluster model contains whole or fragmented side chains of nine active site amino acids surrounding the bound substrates, Glu189, Tyr197, Ser199, Ile384, Leu391, Leu492, Cys493, Arg508, and Val709
additional information
I6LHT8
enzyme structure and structure-based mechanistic models, overview. The QM cluster model contains whole or fragmented side chains of nine active site amino acids surrounding the bound substrates, Glu189, Tyr197, Ser199, Ile384, Leu391, Leu492, Cys493, Arg508, and Val709
additional information
-
enzyme structure and structure-based mechanistic models, overview. The QM cluster model contains whole or fragmented side chains of nine active site amino acids surrounding the bound substrates, Glu189, Tyr197, Ser199, Ile384, Leu391, Leu492, Cys493, Arg508, and Val709
additional information
subunit BSSalpha adopts a conserved Gly radical enzyme fold. The beta subunit BSSbeta adopts a fold similar to that of a high potential iron-sulfur protein HiPIP. Subunit gamma, BSSgamma, is only 19% identical to BSSbeta, but they share the basic HiPIP fold along with the extended beta-sheet and hairpin loop. Analysis of BSSalpha-BSSgamma subunit interactions and of BSSalpha-BSSbeta subunit interactions, global structural changes in the BSSalpha-BSSgamma complex, influence of BSSbeta on the structure and flexibility of BSSalpha, overview
additional information
-
enzyme structure and structure-based mechanistic models, overview. The QM cluster model contains whole or fragmented side chains of nine active site amino acids surrounding the bound substrates, Glu189, Tyr197, Ser199, Ile384, Leu391, Leu492, Cys493, Arg508, and Val709
-
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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).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). A2TBP7_9BACT
251
0
28578
TrEMBL
Mitochondrion (Reliability: 3)
Q39VF1_GEOMG
Geobacter metallireducens (strain ATCC 53774 / DSM 7210 / GS-15)
862
0
97613
TrEMBL
-
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
14500
-
alpha, alpha, beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE
230000
-
determined by gel filtration, consistent with an alpha2beta2gamma2 subunit structure
6500
-
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
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotrimer
hexamer
tetramer
additional information
heterotrimer
1 * 100000 + 1 * 8500 + 1 * 6500, the enzyme consists of three subunits encoded by three different genes and of very different sizes: the large subunit of circa 100 kDa contains the glycyl radical in the active site and presumably carries out the catalysis, whereas the two small subunits of 8.5 and 6.5 kDa each contain a low-potential [4Fe4S]-cluster
heterotrimer
1 * 100000 + 1 * 8500 + 1 * 6500, the enzyme consists of three subunits encoded by three different genes and of very different sizes: the large subunit of circa 100 kDa contains the glycyl radical in the active site and carries out the catalysis
hexamer
-
alpha2 beta2 gamma2, three subunits encoded by the genes tutF, tutD, and tutG
hexamer
-
the structural model for benzylsuccinate synthase proposes an alpha2beta2gamma2-complex
hexamer
-
alpha2 beta2 gamma2, three subunits encoded by the genes tutF, tutD, and tutG
-
tetramer
-
alpha, alpha', beta, gamma, 1 * 97000 + 1 * 97000 + 1 * 14500 + 1 * 6500, SDS-PAGE
tetramer
-
alpha, alpha', beta, gamma, 1 * 94000 + 1 * 90000 + 1 * 12000 + 1 * 10000, SDS-PAGE
additional information
I6LHT8
the enzyme has a trimeric structure of alpha-, beta- and gamma subunits. Structure comparisons of BSS enzymes and BSS-like fumarate-adding enzymes (FAEs)
additional information
-
the enzyme has a trimeric structure of alpha-, beta- and gamma subunits. Structure comparisons of BSS enzymes and BSS-like fumarate-adding enzymes (FAEs)
additional information
the enzyme has a trimeric structure of alpha-, beta- and gamma subunits. Structure comparisons of BSS enzymes and BSS-like fumarate-adding enzymes (FAEs)
additional information
analysis of BSSalpha-BSSgamma subunit interactions and of BSSalpha-BSSbeta subunit interactions, detailed overview
additional information
-
the enzyme has a trimeric structure of alpha-, beta- and gamma subunits. Structure comparisons of BSS enzymes and BSS-like fumarate-adding enzymes (FAEs)
-
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
enzyme BSS needs to be posttranslationally activated to the active, radical-containing state by a separate activating enzyme, which is encoded in a common operon with the genes for the BSS subunits and belongs to the family of S-adenosyl-methionine-dependent radical enzymes
additional information
-
enzyme BSS needs to be posttranslationally activated to the active, radical-containing state by a separate activating enzyme, which is encoded in a common operon with the genes for the BSS subunits and belongs to the family of S-adenosyl-methionine-dependent radical enzymes
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant trimeric enzyme BSS in complex with fumarate and toluene or recombinant dimeric BSSalphagamma enzyme form in complex with fumarate, sitting drop vapor diffusion method, mixing of 8 mg/ml trimeric enzyme in 50 mM Tris, pH 7.6, 15% v/v glycerol, and 200 mM NaCl, in a 2:1 ratio with well solution containing 25% w/v PEG 3350, 100 mM Tris, pH 8.5, 60 mM KCl, and 5 mM fumarate, 0.001-0.002 ml of toluene is added to the bottom of the well and allowed to diffuse slowly into the protein drop, 3 weeks at room temperature. Mixing of 15 mg/ml dimeric enzyme in 20 mM HEPES, pH 7.6, 100 mM NaCl, and 5 mM fumarate, in a 1:1 ratio with well solution containing 20% w/v PEG 400, 50 mM Bis-Tris, pH 6.5, and 25 mM Tris, pH 8.0, several days at room temperature, soaking of crystals in mother liquor with ligands at 50 mM, X-ray diffraction structure determination and analysis at 3.3 A and 2.0 A resolution, respectively, modelling
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C29S
-
tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
C9Y/A10S
-
tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
C29S
-
tutG gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
-
C9Y/A10S
-
tutF gene, strong loss of activity, engineered strain unable to grow with toluene as carbon source
-
E509A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of fumarate
F384A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of toluene
L390A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of toluene
L491A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of toluene
Q706A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of fumarate
S827A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of fumarate
V708A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of toluene
Y829A
Q8L1A3
site-directed mutagenesis, the mutation affects binding of toluene
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
extremely oxygen sensitive
rapidly inactivated by oxygen with a half-life of 20-30 s upon exposure to air
-
210663
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose, hydroxyapatite, anion exchange chromatography, enzyme severely inactivated during purification
-
hydroxyapatite, gel filtration, 95% loss of specific activity after gel filtration
-
recombinant enzyme from Escherichia coli strain BL21(DE3)
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli BL21DE3 cells, the vectors pET-28b, pACYC-Duet, pET-Duet and pRSF-Duet are used
-
gene bssA, phylogenetic analysis
gene order of all known bss operons is bssDCAB, starting with the gene for the activating enzyme and followed by those for the gamma-, alpha- and beta-subunits, phylogenetic analysis
recombinant enzyme expression in Escherichia coli strain BL21(DE3)
gene order of all known bss operons is bssDCAB, starting with the gene for the activating enzyme and followed by those for the gamma-, alpha- and beta-subunits, phylogenetic analysis
I6LHT8
gene order of all known bss operons is bssDCAB, starting with the gene for the activating enzyme and followed by those for the gamma-, alpha- and beta-subunits, phylogenetic analysis
-
gene order of all known bss operons is bssDCAB, starting with the gene for the activating enzyme and followed by those for the gamma-, alpha- and beta-subunits, phylogenetic analysis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the abundance of the enzyme increases in cells growing anaerobically with toluene. Under anoxic conditions, subunit bssA mRNA copy numbers are up to 129fold higher in cells growing with toluene as compared to cells growing with benzoate
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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
environmental protection
additional information
environmental protection
-
toluene is a widespread contaminant and can be degraded under anoxic conditions, catalyzed by benzylsuccinate synthase
environmental protection
-
toluene is a widespread contaminant and can be degraded under anoxic conditions, catalyzed by benzylsuccinate synthase
environmental protection
-
toluene is a widespread contaminant and can be degraded under anoxic conditions, catalyzed by benzylsuccinate synthase
environmental protection
-
toluene is a widespread contaminant and can be degraded under anoxic conditions, catalyzed by benzylsuccinate synthase
environmental protection
enzyme BSS and the growing number of additional fumarate-adding enzymes have become model cases for environmental processes in contaminated soils and deep anoxic subsediment habitats, and their isotopic preferences and conserved sequences serving as templates for molecular probes are employed as tools for monitoring these processes in situ
environmental protection
-
toluene is a widespread contaminant and can be degraded under anoxic conditions, catalyzed by benzylsuccinate synthase
-
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
additional information
-
target for the detection of toluene degrading organisms in environmental samples
-
additional information
-
target for the detection of toluene degrading organisms in environmental samples
-
additional information
-
target for the detection of toluene degrading organisms in environmental samples
-
additional information
-
target for the detection of toluene degrading organisms in environmental samples
-
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Krieger, C.J.; Rosebooms, W.; Albrachts, P.J.; Spormann, A.M.
A stable organic free radical in anaerobic benzylsuccinate synthase of Azoarcus sp. strain T
J. Biol. Chem.
276
12924-12927
2001
Azoarcus sp., Azoarcus sp. T
brenda
Beller, H.R.; Edwards, E.A.
Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture
Appl. Environ. Microbiol.
66
5503-5505
2000
Methanothrix sp., Methanospirillum sp., Desulfotomaculum
brenda
Beller, H.R.; Spormann, A.M.
Substrate range of benzylsuccinate synthase from Azoarcus sp. strain T
FEMS Microbiol. Lett.
178
147-153
1999
Azoarcus sp.
brenda
Leutwein, C.; Heider, J.
Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate
Microbiology
145
3265-3271
1999
Thauera aromatica
brenda
Beller, H.R.; Spormann, A.M.
Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product
J. Bacteriol.
180
5454-5457
1998
Azoarcus sp.
brenda
Leuthner, B.; Leutwein, C.; Schultz,H.; Hrth, P.; Haehnel, W.; Schiltz, E.; Schgger, H.; Heider, J.
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalaysing the first step in anaerobic toluene metabolism
Mol. Microbiol.
28
615-628
1998
Thauera aromatica
brenda
Verfrth, K.; Pierik, A.J.; Leutwein, C.; Zorn, S.; Heider, J.
Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and m-xylene metabolism
Arch. Microbiol.
181
155-162
2004
Azoarcus sp., Thauera aromatica, Azoarcus sp. T, Thauera aromatica K172
brenda
Qiao, C.; Marsh, E.N.G.
Mechanism of benzylsuccinate synthase: Stereochemistry of toluene addition to fumarate and maleate
J. Am. Chem. Soc.
127
8608-8609
2005
Thauera aromatica
brenda
Bhandare, R.; Calabro, M.; Coschigano, P.W.
Site-directed mutagenesis of the Thauera aromatica strain T1 tutE tutFDGH gene cluster
Biochem. Biophys. Res. Commun.
346
992-998
2006
Thauera aromatica, Thauera aromatica T1
brenda
Li, L.; Marsh, E.N.
Deuterium isotope effects in the unusual addition of toluene to fumarate catalyzed by benzylsuccinate synthase
Biochemistry
45
13932-13938
2006
Thauera aromatica
brenda
Winderl, C.; Schaefer, S.; Lueders, T.
Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker
Environ. Microbiol.
9
1035-1046
2007
Thauera aromatica, Azoarcus sp., Geobacter grbiciae, Geobacter sp., Desulfobacula toluolica, Desulfosarcina cetonica, Desulfotomaculum sp., Azoarcus sp. T, Desulfotomaculum sp. OX39, Geobacter sp. TMJ1, Thauera aromatica K172
brenda
Li, L.; Patterson, D.P.; Fox, C.C.; Lin, B.; Coschigano, P.W.; Marsh, E.N.
Subunit structure of benzylsuccinate synthase (dagger)
Biochemistry
48
1284-1292
2009
Thauera aromatica
brenda
Musat, F.; Galushko, A.; Jacob, J.; Widdel, F.; Kube, M.; Reinhardt, R.; Wilkes, H.; Schink, B.; Rabus, R.
Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria
Environ. Microbiol.
11
209-219
2009
uncultured delta proteobacterium
brenda
Vogt, C.; Cyrus, E.; Herklotz, I.; Schlosser, D.; Bahr, A.; Herrmann, S.; Richnow, H.H.; Fischer, A.
Evaluation of toluene degradation pathways by two-dimensional stable isotope fractionation
Environ. Sci. Technol.
42
7793-7800
2008
Thauera aromatica, Azoarcus sp., Blastochloris sulfoviridis, Desulfosarcina cetonica, Azoarcus sp. T
brenda
Salinero, K.; Keller, K.; Feil, W.; Feil, H.; Trong, S.; Di Bartolo, G.; Lapidus, A.
Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: Indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation
BMC Genomics
10
351
2009
no activity in Dechloromonas aromatica RCB
brenda
Weelink, S.A.; van Doesburg, W.; Saia, F.T.; Rijpstra, W.I.; Roeling, W.F.; Smidt, H.; Stams, A.J.
A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor
FEMS Microbiol. Ecol.
70
575-585
2009
Georgfuchsia toluolica, Georgfuchsia toluolica G5G6
brenda
Winderl, C.; Penning, H.; Netzer, F.V.; Meckenstock, R.U.; Lueders, T.
DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment
ISME J.
4
1314-1325
2010
Clostridia
brenda
Hilberg, M.; Pierik, A.J.; Bill, E.; Friedrich, T.; Lippert, M.L.; Heider, J.
Identification of FeS clusters in the glycyl-radical enzyme benzylsuccinate synthase via EPR and Moessbauer spectroscopy
J. Biol. Inorg. Chem.
17
49-56
2012
Escherichia coli
brenda
Bharadwaj, V.S.; Dean, A.M.; Maupin, C.M.
Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study
J. Am. Chem. Soc.
135
12279-12288
2013
Thauera sp. DNT-1 (Q8L1A3)
brenda
Kuemmel, S.; Kuntze, K.; Vogt, C.; Boll, M.; Heider, J.; Richnow, H.H.
Evidence for benzylsuccinate synthase subtypes obtained by using stable isotope tools
J. Bacteriol.
195
4660-4667
2013
Thauera aromatica (O87943), Azoarcus sp. (Q8VPT7), Desulfosarcina cetonica (A2TBP7), Geobacter metallireducens (Q39VF1), Azoarcus sp. T (Q8VPT7), Geobacter metallireducens GS-15 / ATCC 53774 / DSM 7210 (Q39VF1), Thauera aromatica K172 (O87943), Desulfosarcina cetonica DSM 7267 (A2TBP7)
brenda
Szaleniec, M.; Heider, J.
Modeling of the reaction mechanism of enzymatic radical C-C coupling by benzylsuccinate synthase
Int. J. Mol. Sci.
17
514
2016
Thauera aromatica (O87943 AND O87944 AND O87942), Thauera aromatica
brenda
Funk, M.A.; Marsh, E.N.; Drennan, C.L.
Substrate-bound structures of benzylsuccinate synthase reveal how toluene is activated in anaerobic hydrocarbon degradation
J. Biol. Chem.
290
22398-22408
2015
Thauera aromatica (O87943 AND O87944 AND O87942)
brenda
Heider, J.; Szaleniec, M.; Martins, B.M.; Seyhan, D.; Buckel, W.; Golding, B.T.
Structure and function of benzylsuccinate synthase and related fumarate-adding glycyl radical enzymes
J. Mol. Microbiol. Biotechnol.
26
29-44
2016
Thauera aromatica (O87943 AND O87944 AND O87942), Azoarcus sp. CIB (I6LHT8), Desulfosarcina cetonica, Thauera aromatica K172 (O87943 AND O87944 AND O87942)
brenda
Funk, M.A.; Judd, E.T.; Marsh, E.N.; Elliott, S.J.; Drennan, C.L.
Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity
Proc. Natl. Acad. Sci. USA
111
10161-10166
2014
Thauera aromatica (O87943 AND O87944 AND O87942)
brenda
Meyer-Cifuentes, I.; Gruhl, S.; Haange, S.B.; Luensmann, V.; Jehmlich, N.; Bergen, M.V.; Heipieper, H.J.; Mueller, J.A.
Benzylsuccinate synthase is post-transcriptionally regulated in the toluene-degrading denitrifier Magnetospirillum sp. strain 15-1
Microorganisms
8
681
2020
Magnetospirillum sp. 15-1
brenda
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