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Literature summary for 4.2.1.120 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
Search for more literature for 4.2.1.120

Cloned(Commentary)

Cloned (Comment) Organism
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

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

Metals/Ions

Metals/Ions Comment Organism Structure
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)

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

Organism

Organism UniProt Comment Textmining
Clostridium aminobutyricum P55792
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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]

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-aminobutyryl-CoA the natural substrate is 4-aminobutyrate Clostridium aminobutyricum ? + H2O
-
 r
4-hydroxybutyryl-CoA
-
 Clostridium aminobutyricum crotonyl-CoA + H2O
-
 r
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

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

Synonyms

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

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
FAD one FAD per enzyme subunit, FAD contents of enzyme mutants compared to the wild-type, overview Clostridium aminobutyricum
[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

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