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Information on EC 4.2.1.150 - short-chain-enoyl-CoA hydratase and Organism(s) Clostridium acetobutylicum and UniProt Accession P52046

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.150 short-chain-enoyl-CoA hydratase
IUBMB Comments
The enzyme from the bacterium Clostridium acetobutylicum is part of the central fermentation pathway and plays a key role in the production of both acids and solvents. It is specific for short, C4-C6, chain length substrates and exhibits an extremely high turnover number for crotonyl-CoA. cf. EC 4.2.1.17, enoyl-CoA hydratase and EC 4.2.1.74, long-chain-enoyl-CoA hydratase.
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Clostridium acetobutylicum
UNIPROT: P52046
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The taxonomic range for the selected organisms is: Clostridium acetobutylicum
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
chromodomain y-like protein, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3-hydroxybutyryl-CoA dehydratase
-
-
L-3-hydroxyacyl-CoA hydro-lyase
-
-
additional information
-
cf. EC 4.2.1.17
PATHWAY SOURCE
PATHWAYS
-
-, -, -, -, -, -, -, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
short-chain-(3S)-3-hydroxyacyl-CoA hydro-lyase
The enzyme from the bacterium Clostridium acetobutylicum is part of the central fermentation pathway and plays a key role in the production of both acids and solvents. It is specific for short, C4-C6, chain length substrates and exhibits an extremely high turnover number for crotonyl-CoA. cf. EC 4.2.1.17, enoyl-CoA hydratase and EC 4.2.1.74, long-chain-enoyl-CoA hydratase.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-hydroxybutyryl-CoA
crotonyl-CoA + H2O
show the reaction diagram
-
-
-
r
3-hydroxybutyryl-CoA
crotonyl-CoA + H2O
show the reaction diagram
-
-
-
-
?
a short-chain trans-2-enoyl-CoA + H2O
short-chain (3S)-3-hydroxyacyl-CoA
show the reaction diagram
-
the enzyme is specific for short chain fatty acyl-CoA substrates and is sensitive to high concentrations of crotonyl-CoA. It requires a complete coenzyme A thioester substrate for efficient catalysis
-
-
?
crotonyl-CoA + H2O
3-hydroxybutyryl-CoA
show the reaction diagram
-
-
-
-
?
crotonyl-CoA + H2O
?
show the reaction diagram
-
-
-
-
?
hexenoyl-CoA + H2O
?
show the reaction diagram
additional information
?
-
enzyme residues Ser69 and Ala24 are signature residues of CaCRT, resulting in a distinct ADP binding mode wherein the ADP moiety of acetoacetyl-CoA is bound at a different position compared with other crotonases. The substrate specificity of crotonase enzymes is determined by both the structural feature of the a3 helix region and the residues contributing the enoyl-CoA binding pocket. A tight formed a3 helix and two phenylalanine residues, Phe143 and Phe233, aid CaCRT to accommodate crotonyl-CoA as the substrate. Phe143 and Phe233 are key residues for the constitution of the crotonyl binding pocket to accommodate the four-carbon crotonyl-CoA as a substrate
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
3-hydroxybutyryl-CoA
crotonyl-CoA + H2O
show the reaction diagram
-
-
-
-
?
crotonyl-CoA + H2O
3-hydroxybutyryl-CoA
show the reaction diagram
-
-
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
crotonyl-CoA
additional information
-
not inhibited by hexenoyl-CoA
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
crotonyl-CoA
0.13
hexenoyl-CoA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6155
-
25°C, pH 7.5
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
the enzyme catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway, molecular mechanism
additional information
substrate binding pocket structure and mechanism, overview. CaCRT uses a unique CoA binding mode. The Ser69 residue in CaCRT is hydrogen-bonded with N6 of AcAc-CoA, in contrast to the corresponding Lys101 and Val74 residues in ECH and DmdD, and is involved in the stabilization of the adenine ring. Moreover, Ala24 of CaCRT is located near the phosphate moiety, whereas the corresponding Lys31 residue of DmdD is hydrogen-bonded with this moiety
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
158000
28200
-
x * 28200, calculated from amino acid sequence
29000
-
x * 29000, SDS-PAGE
40000
-
4 * 40000, SDS-PAGE
43000
-
4 * 43000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
a dimer of trimers
homotetramer
-
4 * 40000, SDS-PAGE
tetramer
-
4 * 43000, SDS-PAGE
additional information
the CaCRT monomer consists of an N-terminal (NTD) and a C-terminal domain (CTD). The NTD (beta1-beta7 and alpha1-alpha9) harbors the canonical crotonase fold, where a large beta-sheet (beta1-beta4 and beta6) is organized with a small beta-sheet (beta5 and beta7) forming two perpendicular beta-sheets. The CTD consists of three alpha-helices (alpha10-alpha12), and this domain mediates the oligomerization of CaCRT. Additionally, the extended alpha-helix (alpha12) interacts with the NTD of a neighboring monomer and participates in the formation of its substrate binding site. The CTDs of six monomers participate mainly in the formation of the hexameric interface
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged enzyme in apo- and acetoacetyl-CoA bound forms, from precipitant solution containing 30% PEG 400, 0.1 M sodium cacodylate, pH 6.5, and 0.2 M lithium sulfate, 22°C, 5 days, X-ray diffraction structure determination and analysis at 2.0-2.2 A resolution, molecular replacement and modelling
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F143A
site-directed mutagenesis, almost inactive mutant
F233A
site-directed mutagenesis, almost inactive mutant
F82A
site-directed mutagenesis, the mutant shows similar activity compared to wild-type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
lyophilization results in negligible loss of enzymatic activity
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4 or -20°C, Tris-HCl, pH 8.0, rapid loss of activity
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain B834 by nickel affinity chromatography and gel filtration
acetone precipitation, Sephadex G-200 gel filtration, and DEAE-Sephadex column chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
the CaCRT coding gene (Met1-Arg261) is amplified by PCR using the chromosomal DNA of Clostridium acetobutylicum strain ATCC 824 as a template, recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain B834
expressed in Escherichia coli XL1-Blue cells
-
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Waterson, R.M.; Conway, R.S.
Enoyl-CoA hydratases from Clostridium acetobutylicum and Escherichia coli
Methods Enzymol.
71
421-430
1981
Clostridium acetobutylicum
Manually annotated by BRENDA team
Waterson, R.M.; Castellino, F.J.; Hass, G.M.; Hill, R.L.
Purification and characterization of cortonase from Clostridium acetobutylicum
J. Biol. Chem.
247
5266-5271
1972
Clostridium acetobutylicum
Manually annotated by BRENDA team
Boynton, Z.L.; Bennett, G.N.; Rudolph, F.B.
Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824
J. Bacteriol.
178
3015-3024
1996
Clostridium acetobutylicum
Manually annotated by BRENDA team
Willadsen, P.; Buckel, W.
Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum
FEMS Microbiol. Lett.
70
187-191
1990
Clostridium acetobutylicum
Manually annotated by BRENDA team
Kim, E.J.; Kim, Y.J.; Kim, K.J.
Structural insights into substrate specificity of crotonase from the n-butanol producing bacterium Clostridium acetobutylicum
Biochem. Biophys. Res. Commun.
451
431-435
2014
Clostridium acetobutylicum (P52046), Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 (P52046)
Manually annotated by BRENDA team