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Literature summary extracted from

  • Zhang, J.; Friedrich, P.; Pierik, A.J.; Martins, B.M.; Buckel, W.
    Substrate-induced radical formation in 4-hydroxybutyryl coenzyme A dehydratase from Clostridium aminobutyricum (2015), Appl. Environ. Microbiol., 81, 1071-1084 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.1.120 gene abfD, recombinant expression of C-terminally Strep-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), method evaluation and optimization, addition of Fe is required Clostridium aminobutyricum

Protein Variants

EC Number Protein Variants Comment Organism
4.2.1.120 A460G site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 C103A site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type Clostridium aminobutyricum
4.2.1.120 C299A site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type Clostridium aminobutyricum
4.2.1.120 C99A site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type Clostridium aminobutyricum
4.2.1.120 E257Q site-directed mutagenesis, inactive mutant forming tetramers Clostridium aminobutyricum
4.2.1.120 E455Q site-directed mutagenesis, inactive mutant forming tetramers Clostridium aminobutyricum
4.2.1.120 H292C site-directed mutagenesis of a catalytic residue, inactive mutant with dimeric or tetrameric subunit composition compared to wild-type Clostridium aminobutyricum
4.2.1.120 H292E site-directed mutagenesis of a catalytic residue, inactive mutant with tetrameric subunit composition compared to wild-type Clostridium aminobutyricum
4.2.1.120 K300Q site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 M149S site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 additional information iron and FAD contents of enzyme mutants compared to the wild-type, mutant phenotypes, overview Clostridium aminobutyricum
4.2.1.120 Q101E site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 R90N site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 T190V site-directed mutagenesis, almost inactive mutant forming tetramers Clostridium aminobutyricum
4.2.1.120 T190V site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers Clostridium aminobutyricum
4.2.1.120 Y296F site-directed mutagenesis, almost inactive mutant forming tetramers Clostridium aminobutyricum
4.2.1.120 Y296W site-directed mutagenesis, inactive mutant Clostridium aminobutyricum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.1.120 Fe2+ non-heme iron in a [4Fe-4S]-center, iron and FAD contents of enzyme mutants compared to the wild-type, non-heme iron is quantified with the iron chelator 3-(2-pyridyl)-5,6-bis(5-sulfo-2-furyl)-1,2,4-triazine (disodium salt trihydrate), overview Clostridium aminobutyricum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.1.120 4-aminobutyryl-CoA Clostridium aminobutyricum the natural substrate is 4-aminobutyrate ? + H2O
-
 r

Organism

EC Number Organism UniProt Comment Textmining
4.2.1.120 Clostridium aminobutyricum P55792
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.1.120 recombinant C-terminally Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography Clostridium aminobutyricum

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.1.120 4-hydroxybutanoyl-CoA = (E)-but-2-enoyl-CoA + H2O substrate-induced radical formation in 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The conversion of 4-hydroxybutyryl-CoA to crotonyl-CoA involves the abstraction of the 2Re and 3Si protons. The FAD semiquinone rather than the FAD quinone oxidizes the enolate (or, in the reverse direction, the dienolate) to the enoxy radical (dienoxy radical). The FADH- anion formed, in combination with the T190/E257 dyad, probably acts as a more efficient base to remove the 3Si proton. Reaction mechanism with amino acids proposed to be involved, overview. The release of H2O from Fe1 of the [4Fe-4S]2+ cluster can be facilitated by reduction to [4Fe-4S]+ Clostridium aminobutyricum
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.1.120 2.2
-
 purified recombinant wild-type enzyme, pH 7.4, 22°C Clostridium aminobutyricum
4.2.1.120 4.5
-
 purified recombinant wild-type enzyme reconstituted with FeCl3 and Na2S, pH 7.4, 22°C Clostridium aminobutyricum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.1.120 4-aminobutyryl-CoA the natural substrate is 4-aminobutyrate Clostridium aminobutyricum ? + H2O
-
 r
4.2.1.120 4-hydroxybutyryl-CoA
-
 Clostridium aminobutyricum crotonyl-CoA + H2O
-
 r
4.2.1.120 additional information 4-hydroxybutyryl-CoA dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Specific activity of enzyme 4HBD is measured anaerobically in a coupled assay based on determining the amount of crotonyl-CoA formed by beta-oxidation to acetyl-CoA Clostridium aminobutyricum ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.2.1.120 homotetramer wild-type enzyme Clostridium aminobutyricum
4.2.1.120 More some enzyme mutant show altered subunit compositions, overview Clostridium aminobutyricum

Synonyms

EC Number Synonyms Comment Organism
4.2.1.120 4-hydroxybutyryl-CoA dehydratase
-
 Clostridium aminobutyricum
4.2.1.120 4-hydroxybutyryl-coenzyme A dehydratase
-
 Clostridium aminobutyricum
4.2.1.120 4HBD
-
 Clostridium aminobutyricum
4.2.1.120 AbfD
-
 Clostridium aminobutyricum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.1.120 22
-
 assay at room temperature Clostridium aminobutyricum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.1.120 7.4
-
 assay at Clostridium aminobutyricum

Cofactor

EC Number Cofactor Comment Organism Structure
4.2.1.120 FAD one FAD per enzyme subunit, FAD contents of enzyme mutants compared to the wild-type, overview Clostridium aminobutyricum
4.2.1.120 [4Fe-4S]-center the enzyme contains one [4Fe-4S]2x02 cluster per enzyme tetramer, reconstitution of the [4Fe-4S] cluster in the purified enzyme with FeCl3 and Na2S Clostridium aminobutyricum

General Information

EC Number General Information Comment Organism
4.2.1.120 additional information kinetic isotope effects with regiospecifically 2H-labeled 4-hydroxybutyrates and isolated enzyme 4HBD Clostridium aminobutyricum