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 | 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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Clostridium aminobutyricum | P55792 | - |
- |
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 | 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 |
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 | 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 | Comment | Organism |
---|---|---|
homotetramer | wild-type enzyme | Clostridium aminobutyricum |
More | some enzyme mutant show altered subunit compositions, overview | Clostridium aminobutyricum |
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 Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Clostridium aminobutyricum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Clostridium aminobutyricum |
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 | Comment | Organism |
---|---|---|
additional information | kinetic isotope effects with regiospecifically 2H-labeled 4-hydroxybutyrates and isolated enzyme 4HBD | Clostridium aminobutyricum |