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Literature summary extracted from

  • Funk, M.A.; Marsh, E.N.; Drennan, C.L.
    Substrate-bound structures of benzylsuccinate synthase reveal how toluene is activated in anaerobic hydrocarbon degradation (2015), J. Biol. Chem., 290, 22398-22408 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.99.11 recombinant enzyme expression in Escherichia coli strain BL21(DE3) Thauera aromatica

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.1.99.11 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 Thauera aromatica

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.1.99.11 toluene + fumarate Thauera aromatica
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 benzylsuccinate
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 ?

Organism

EC Number Organism UniProt Comment Textmining
4.1.99.11 Thauera aromatica O87943 AND O87944 AND O87942 alpha-, beta-, and gamma-subunit
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.1.99.11 recombinant enzyme from Escherichia coli strain BL21(DE3) Thauera aromatica

Reaction

EC Number Reaction Comment Organism Reaction ID
4.1.99.11 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 Thauera aromatica
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.99.11 additional information 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 Thauera aromatica ?
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 ?
4.1.99.11 toluene + fumarate
-
 Thauera aromatica benzylsuccinate
-
 ?
4.1.99.11 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. Both substrates can bind to the BSSalphabetagamma complex Thauera aromatica benzylsuccinate
-
 ?

Subunits

EC Number Subunits Comment Organism
4.1.99.11 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 Thauera aromatica

Synonyms

EC Number Synonyms Comment Organism
4.1.99.11 BSS
-
 Thauera aromatica

Cofactor

EC Number Cofactor Comment Organism Structure
4.1.99.11 glycyl radical the C-C bond-forming reaction performed by BSS and its relatives requires an oxygen-sensitive radical cofactor, mechanism, overview Thauera aromatica

General Information

EC Number General Information Comment Organism
4.1.99.11 evolution enzyme BSS is a member of the glycyl radical enzyme (GRE) family Thauera aromatica
4.1.99.11 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 Thauera aromatica