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Information on EC 4.2.1.120 - 4-hydroxybutanoyl-CoA dehydratase and Organism(s) Clostridium aminobutyricum and UniProt Accession P55792
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4.2.1.120 4-hydroxybutanoyl-CoA dehydrataseIUBMB Comments
Contains FAD and a [4Fe-4S] iron-sulfur cluster. The enzyme has been characterized from several microorganisms, including Clostridium kluyveri, where it participates in succinate fermentation [1,2], Clostridium aminobutyricum, where it participates in 4-aminobutyrate degradation [3,4], and Metallosphaera sedula, where it participates in the 3-hydroxypropionate/4-hydroxybutyrate cycle, an autotrophic CO2 fixation pathway found in some thermoacidophilic archaea .
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Word Map
- 4.2.1.120
- crotonyl-coa
- aminobutyricum
- 4-hydroxybutyrate
- succinyl-coa
-
3-hydroxypropionate/4-hydroxybutyrate
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2-hydroxyacyl-coa
- sedula
- metallosphaera
-
chemolithoautotrophic
-
non-redox
- thermoproteales
-
ignicoccus
-
autotrophy
-
thermoproteus
- kluyveri
- hospitalis
- neutrophilus
- sulfolobales
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dicarboxylate/4-hydroxybutyrate
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hexacyanoferrateiii
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ketyl
- crenarchaeota
The taxonomic range for the selected organisms is: Clostridium aminobutyricum
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
4-hydroxybutyryl-coa dehydratase, 4-hydroxybutyryl-coenzyme a dehydratase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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]+
4-hydroxybutanoyl-CoA = (E)-but-2-enoyl-CoA + H2O
cleavage is achieved by a FAD-dependent oxidation of 4-hydroxybutanoyl-CoA to 4-hydroxycrotonyl-CoA. In a second step, the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2' reaction leading to vinylacetyl-CoA. Isomerization yields crotonyl-CoA
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4-hydroxybutanoyl-CoA = (E)-but-2-enoyl-CoA + H2O
mechanism includes a direct dehydration of of 4-hydroxybutanoyl-CoA to vinylacetyl-CoA
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4-hydroxybutanoyl-CoA = (E)-but-2-enoyl-CoA + H2O
mechanism involves transient one-electron oxidation of the substrate to activate the beta-C-H-bond. the 4Fe-4S-center could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group
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4-hydroxybutanoyl-CoA = (E)-but-2-enoyl-CoA + H2O
the pro-(S) hydrogen atom is stereospecifically abstracted from C-3 of 4-hydroxybutanoyl-CoA, and this atom is not returned to C-4
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PATHWAY SOURCE
PATHWAYS
KEGG
-
-, -, -, -
SYSTEMATIC NAME
IUBMB Comments
4-hydroxybutanoyl-CoA hydro-lyase
Contains FAD and a [4Fe-4S] iron-sulfur cluster. The enzyme has been characterized from several microorganisms, including Clostridium kluyveri, where it participates in succinate fermentation [1,2], Clostridium aminobutyricum, where it participates in 4-aminobutyrate degradation [3,4], and Metallosphaera sedula, where it participates in the 3-hydroxypropionate/4-hydroxybutyrate cycle, an autotrophic CO2 fixation pathway found in some thermoacidophilic archaea [5].
CAS REGISTRY NUMBER
COMMENTARY
129430-44-0
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-aminobutyryl-CoA
? + H2O
the natural substrate is 4-aminobutyrate
-
-
r
4-hydroxybutanoyl-CoA
but-3-enoyl-CoA + H2O
-
reaction involves cleavage of an unactivated C-H bond at the beta-carbon
-
-
r
4-hydroxybutanoyl-CoA
but-3-enoyl-CoA + H2O
-
selective removal of the (2Re)-hydrogen atom. The stereochemical course at C2 and C3 can be described as anti elimination of the two hydrogen atoms, which is identical to that of acyl-CoA dehydrogenases. The formation of the methyl group of crotonyl-CoA from the hydroxymethyl group of 4-hydroxybutanoyl-CoA occurs with retention of configuration
-
-
?
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
-
-
?
additional information
?
-
-
no substrate: cyclopropylcarboxyl-CoA
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-aminobutyryl-CoA
? + H2O
the natural substrate is 4-aminobutyrate
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4Fe-4S-center
a [4Fe-4S]2+ cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide moiety
[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
FAD
a [4Fe-4S]2+cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide moiety
FAD
one FAD per enzyme subunit, FAD contents of enzyme mutants compared to the wild-type, overview
4Fe-4S-center
-
enzyme shows [4Fe-4S]2+ clusters, two clusters/homotetramer. The four iron atoms in each cluster are coordinated in an identical fashion, and there is no direct interaction with substrates. The Fe-S clusters serve a structural rather than a catalytic role in 4-hydroxybutyryl-CoA dehydratase
4Fe-4S-center
-
Fe-S-cluster is difficult to reduce. No equilibration of electrons between the flavin and the Fe-S-center
FAD
-
protein-bound FAD, is easily reduced to the semiquinone and only slowly to the hydroquinone. No equilibration of electrons between the flavin and the Fe-S-center
FAD
-
substrate interacts with the flavin. Partial reduction of the enzyme using dithionite results in formation of a neutral flavin semiquinone, which may interact with the 4Fe-4S-center
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
Iron
Iron
-
16 mol per mol of enzyme, plus 14 mol sulfur per mol of enzyme
Iron
-
enzyme shows [4Fe-4S]2+ clusters, two clusters/homotetramer. The four iron atoms in each cluster are coordinated in an identical fashion, and there is no direct interaction with substrates. The Fe-S clusters serve a structural rather than a catalytic role in 4-hydroxybutyryl-CoA dehydratase
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
enzyme is not inactivated by 5 mM 4-nitrophenol, 5 mM chloramphenicol, and 5 mM hydroxylamine
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
enzyme does not require ATP, MgCl2, and Ti(III)citrate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5
purified recombinant wild-type enzyme reconstituted with FeCl3 and Na2S, pH 7.4, 22°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
enzyme is largely membrane- or particle-bound and may be solubilized by detergent
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
kinetic isotope effects with regiospecifically 2H-labeled 4-hydroxybutyrates and isolated enzyme 4HBD
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). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
some enzyme mutant show altered subunit compositions, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
to 1.6 A resolution. A [4Fe-4S]2+ cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide moiety. The substrate can be bound between the [4Fe-4S]2+ cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversio
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A460G
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
C103A
site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type
C299A
site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type
C99A
site-directed mutagenesis of a catalytic residue, inactive mutant with monomeric, dimeric, or tetrameric subunit composition compared to wild-type
E257Q
site-directed mutagenesis, inactive mutant forming tetramers
E455Q
site-directed mutagenesis, inactive mutant forming tetramers
H292C
site-directed mutagenesis of a catalytic residue, inactive mutant with dimeric or tetrameric subunit composition compared to wild-type
H292E
site-directed mutagenesis of a catalytic residue, inactive mutant with tetrameric subunit composition compared to wild-type
K300Q
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
M149S
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
Q101E
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
R90N
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
T190V
Y296F
site-directed mutagenesis, almost inactive mutant forming tetramers
additional information
iron and FAD contents of enzyme mutants compared to the wild-type, mutant phenotypes, overview
T190V
site-directed mutagenesis, almost inactive mutant forming tetramers
T190V
site-directed mutagenesis, the mutant shows highly reduced enzyme activity and forms tetramers
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
exposure to air results in initial activation followed by irreversible inactivation
-
2966
upon exposure to air at 0°C, complete loss of dehydration activity within 40 min
-
696186
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant C-terminally Strep-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Scherf, U.; Buckel, W.
Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA DELTA3-DELTA2-isomerase from Clostridium aminobutyricum
Eur. J. Biochem.
215
421-429
1993
Clostridium aminobutyricum
Friedrich, P.; Darley, D.J.; Golding, B.T.; Buckel, W.
The complete stereochemistry of the enzymatic dehydration of 4-hydroxybutyryl coenzyme A to crotonyl coenzyme A
Angew. Chem.
47
3254-3257
2008
Clostridium aminobutyricum
Gerhardt, A.; Cinkaya, I.; Linder, D.; Huisman, G.; Buckel, W.
Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA
Arch. Microbiol.
174
189-199
2000
Clostridium aminobutyricum
Mueh, U.; Cinkaya, I.; Albracht, S.P.J.; Buckel, W.
4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: Characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction
Biochemistry
35
11710-11718
1996
Clostridium aminobutyricum
Cinkaya, I.; Buckel, W.; Medina, M.; Gomez-Moreno, C.; Cammack, R.
Electron-nuclear double resonance spectroscopy investigation of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: Comparison with other flavin radical enzymes
Biol. Chem.
378
843-849
1997
Clostridium aminobutyricum
Scott, R.; Naeser, U.; Friedrich, P.; Selmer, T.; Buckel, W.; Golding, B.T.
Stereochemistry of hydrogen removal from the unactivated' C-3 position of 4-hydroxybutyryl-CoA catalysed by 4-hydroxybutyryl-CoA dehydratase
Chem. Commun. (Camb. )
2004
1210-1211
2004
Clostridium aminobutyricum
Muh, U.; Buckel, W.; Bill, E.
Moessbauer study of 4-hydroxybutyryl-CoA dehydratase. Probing the role of an iron-sulfur cluster in an overall non-redox reaction
Eur. J. Biochem.
248
380-384
1997
Clostridium aminobutyricum
Willadsen, P.; Buckel, W.
Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum
FEMS Microbiol. Lett.
70
187-191
1990
Clostridium aminobutyricum
Martins, B.M.; Dobbek, H.; Cinkaya, I.; Buckel, W.; Messerschmidt, A.
Crystal structure of 4-hydroxybutyryl-CoA dehydratase: Radical catalysis involving a [4Fe-4S] cluster and flavin
Proc. Natl. Acad. Sci. USA
101
15645-15649
2004
Clostridium aminobutyricum (P55792), Clostridium aminobutyricum
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