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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The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
a short-chain (3S)-3-hydroxyacyl-CoA = a short-chain trans-2-enoyl-CoA + H2O
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a short-chain (3S)-3-hydroxyacyl-CoA = a short-chain trans-2-enoyl-CoA + H2O
the Mr-Crt catalytic mechanism constitutes a concerted attack by the two glutamate residues. While Glu143 of Mr-Crt protonates the substrate, Glu123 abstracts a proton from a bound water molecule. The Gly120 activates the substrate by a hydrogen bond to the oxygen of the enoyl moiety of the CoA ester. The binding pocket for the CoA moiety is formed by characteristic hydrophobic amino acids and lysine residues
a short-chain (3S)-3-hydroxyacyl-CoA = a short-chain trans-2-enoyl-CoA + H2O
the Mr-Crt catalytic mechanism constitutes a concerted attack by the two glutamate residues. While Glu143 of Mr-Crt protonates the substrate, Glu123 abstracts a proton from a bound water molecule. The Gly120 activates the substrate by a hydrogen bond to the oxygen of the enoyl moiety of the CoA ester. The binding pocket for the CoA moiety is formed by characteristic hydrophobic amino acids and lysine residues
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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.
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
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
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
steady-state kinetics of the CDYL-catalyzed hydratation reaction compared to mitochondrial metabolic enzyme enoyl-CoA hydratase (ECH, EC 4.2.1.17) as a positive control
the C-terminal CoAP domain of the CDY family proteins including CDYL has a three-dimensional structure closely resembling enoyl-CoA hydratase, which catalyzes the hydratation of 2-trans-enoyl-CoA into beta-hydroxyacyl-CoA in mitochondria during beta-oxidation of fatty acids
the C-terminal CoAP domain of the CDY family proteins including CDYL has a three-dimensional structure closely resembling enoyl-CoA hydratase, which catalyzes the hydratation of 2-trans-enoyl-CoA into beta-hydroxyacyl-CoA in mitochondria during beta-oxidation of fatty acids
the C-terminal CoAP domain of the CDY family proteins including CDYL has a three-dimensional structure closely resembling enoyl-CoA hydratase, which catalyzes the hydratation of 2-trans-enoyl-CoA into beta-hydroxyacyl-CoA in mitochondria during beta-oxidation of fatty acids
Cdyl knockout mice are embryonically lethal or died shortly after birth with significantly reduced levels of histone Kcr in elongating spermatids from Cdyl transgenic mice compared to that from wild-type mice, although the level of histone acetylation is comparable
the levels of total histone Kcr and H2BK12cr on the promoter of known CDYL target genes BDNF, NEUROD1, SCG10, and MYT1 increase significantly in CDYL-KO HeLa cells, whereas the regional level of H3K27me3 in these cells decreases, expression patterns, overview
Cdyl knockout mice are embryonically lethal or died shortly after birth with significantly reduced levels of histone Kcr in elongating spermatids from Cdyl transgenic mice compared to that from wild-type mice, although the level of histone acetylation is comparable
the chromodomain Y-like protein CDYL acts as a crotonyl-CoA hydratase to negatively regulate histone crotonylation. The chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to beta-hydroxybutyryl-CoA. This activity is intrinsically linked to the transcription repression function of CDYL and is implemented in reactivation of sex chromosome-linked genes and histone replacement during spermatogenesis. The negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids
the chromodomain Y-like protein CDYL acts as a crotonyl-CoA hydratase to negatively regulate histone crotonylation. The chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to beta-hydroxybutyryl-CoA. This activity is intrinsically linked to the transcription repression function of CDYL and is implemented in reactivation of sex chromosome-linked genes and histone replacement during spermatogenesis. The negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Cdyl regulates the expression of sex chromosome-linked escaped genes in postmeiotic spermatogenic cells by mainly influencing histone Kcr on the gene promoters. The enzyme is important in the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (azoospermia factor c)-deleted infertile men
the chromodomain Y-like protein CDYL acts as a crotonyl-CoA hydratase to negatively regulate histone crotonylation. The chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to beta-hydroxybutyryl-CoA. This activity is intrinsically linked to the transcription repression function of CDYL and is implemented in reactivation of sex chromosome-linked genes and histone replacement during spermatogenesis. The negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids
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
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
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
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
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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
generation of Cdyl transgenic mice by microinjection of a Cdyl construct into the pro-nuclei of fertilized oocytes, derived from intercross of C57BL/6 3 CBA F1 mice
generation of Cdyl transgenic mice by microinjection of a Cdyl construct into the pro-nuclei of fertilized oocytes, derived from intercross of C57BL/6 3 CBA F1 mice
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain B834 by nickel affinity chromatography and gel filtration
gene Mrub_2284, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis, recombinant expression of C-terminally His-tagged enzyme in Escherichia coli strain Rosetta (DE3), subcloning in Escherichia coli strain XL-1 Blue
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
Purification and properties of an iron-sulfur and FAD-containing 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA DELTA3-DELTA2-isomerase from Clostridium aminobutyricum
Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824
Conversion of 4-hydroxybutyrate to acetyl coenzyme A and its anapleurosis in the Metallosphaera sedula 3-hydroxypropionate/4-hydroxybutyrate carbon fixation pathway