Cloned (Comment) | Organism |
---|---|
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 | Clostridium acetobutylicum |
Crystallization (Comment) | Organism |
---|---|
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 | Clostridium acetobutylicum |
Protein Variants | Comment | Organism |
---|---|---|
F143A | site-directed mutagenesis, almost inactive mutant | Clostridium acetobutylicum |
F233A | site-directed mutagenesis, almost inactive mutant | Clostridium acetobutylicum |
F82A | site-directed mutagenesis, the mutant shows similar activity compared to wild-type enzyme | Clostridium acetobutylicum |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Clostridium acetobutylicum | P52046 | - |
- |
Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 | P52046 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain B834 by nickel affinity chromatography and gel filtration | Clostridium acetobutylicum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-hydroxybutyryl-CoA | - |
Clostridium acetobutylicum | crotonyl-CoA + H2O | - |
r | |
3-hydroxybutyryl-CoA | - |
Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 | crotonyl-CoA + H2O | - |
r | |
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 | Clostridium acetobutylicum | ? | - |
? | |
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 | Clostridium acetobutylicum ATCC 824 / DSM 792 / JCM 1419 / LMG 5710 / VKM B-1787 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
hexamer | a dimer of trimers | Clostridium acetobutylicum |
More | 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 | Clostridium acetobutylicum |
Synonyms | Comment | Organism |
---|---|---|
CaCRT | - |
Clostridium acetobutylicum |
crotonase | - |
Clostridium acetobutylicum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Clostridium acetobutylicum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Clostridium acetobutylicum |
General Information | Comment | Organism |
---|---|---|
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 | Clostridium acetobutylicum |
physiological function | the enzyme catalyzes the dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA in the n-butanol biosynthetic pathway, molecular mechanism | Clostridium acetobutylicum |