EC Number | Cloned (Comment) | Organism |
---|---|---|
2.3.1.9 | recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3) | Cupriavidus necator |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.3.1.9 | purified recombinant enzyme in apoform or with bound CoA, hanging drop vapor diffusion method, mixing 0.0012 ml of 25 mg/ml protein in 40 mM Tris-HCl, pH 8.0, with 0.0012 ml of reservoir solution containing 17% PEG 8000, 0.1 M HEPES pH 7.0, and equilibration against 0.5 ml of reservoir solution, 20-22°C, 7 days, X-ray diffraction structure determination and analysis at 1.4-1.5 A resolution, molecular replacement method using the structure of Mycobacterium tuberculosis thiolase MtFadA5, PDB ID 4UBU as a search model, structure modeling | Cupriavidus necator |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.3.1.9 | C378A | site-directed mutagenesis, almost inactive mutant | Cupriavidus necator |
2.3.1.9 | C91A | site-directed mutagenesis, almost inactive mutant | Cupriavidus necator |
2.3.1.9 | H348A | site-directed mutagenesis, almost inactive mutant | Cupriavidus necator |
2.3.1.9 | K18A | site-directed mutagenesis, the mutant shows over 80% reduced activity compared to the wild-type enzyme | Cupriavidus necator |
2.3.1.9 | K217A | site-directed mutagenesis, the mutant shows 60% reduced activity compared to the wild-type enzyme | Cupriavidus necator |
2.3.1.9 | Q151A | site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme | Cupriavidus necator |
2.3.1.9 | R210A | site-directed mutagenesis, the mutant shows 70% reduced activity compared to the wild-type enzyme | Cupriavidus necator |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.3.1.9 | 2 acetyl-CoA | Cupriavidus necator | - |
CoA + acetoacetyl-CoA | - |
r | |
2.3.1.9 | 2 acetyl-CoA | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | - |
CoA + acetoacetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | Cupriavidus necator | - |
2 acetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | - |
2 acetyl-CoA | - |
r | |
2.3.1.9 | additional information | Cupriavidus necator | the enzyme shows degradative thiolase activity by converting 3-oxoacyl-CoA to acyl-CoA | ? | - |
? | |
2.3.1.9 | additional information | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | the enzyme shows degradative thiolase activity by converting 3-oxoacyl-CoA to acyl-CoA | ? | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.3.1.9 | Cupriavidus necator | Q0KAI3 | gene H16_A1887; gene H16_A1887 | - |
2.3.1.9 | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | Q0KAI3 | gene H16_A1887; gene H16_A1887 | - |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.3.1.9 | recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) to about 95% purity by nickel affinity chromatography and gel filtration | Cupriavidus necator |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.3.1.9 | 2 acetyl-CoA | - |
Cupriavidus necator | CoA + acetoacetyl-CoA | - |
r | |
2.3.1.9 | 2 acetyl-CoA | - |
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | CoA + acetoacetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | - |
Cupriavidus necator | 2 acetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | CoA binding mode, overview | Cupriavidus necator | 2 acetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | - |
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | 2 acetyl-CoA | - |
r | |
2.3.1.9 | CoA + acetoacetyl-CoA | CoA binding mode, overview | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | 2 acetyl-CoA | - |
r | |
2.3.1.9 | additional information | the enzyme shows degradative thiolase activity by converting 3-oxoacyl-CoA to acyl-CoA | Cupriavidus necator | ? | - |
? | |
2.3.1.9 | additional information | the enzyme shows degradative thiolase activity by converting 3-oxoacyl-CoA to acyl-CoA | Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 | ? | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.3.1.9 | homodimer | enzyme ReH16_A1887 exists as a dimer both in the crystal and in solution. The interaction between alpha3 and alpha5 from the other chain also mediates the dimerization through hydrophobic interactions with residues Glu30, Leu74, Val71, Ile192, Ser139, Met140, Arg143 and Tyr 144 | Cupriavidus necator |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.3.1.9 | 3-ketoacyl-CoA thiolase | - |
Cupriavidus necator |
2.3.1.9 | A1887 | - |
Cupriavidus necator |
2.3.1.9 | ACAT | - |
Cupriavidus necator |
2.3.1.9 | acetoacetyl-CoA thiolase | - |
Cupriavidus necator |
2.3.1.9 | KACT | - |
Cupriavidus necator |
2.3.1.9 | type II thiolase | - |
Cupriavidus necator |
EC Number | General Information | Comment | Organism |
---|---|---|---|
2.3.1.9 | metabolism | the enzyme shows degradative thiolase activity catalyzing the fourth step of beta-oxidation degradative pathways by converting 3-oxoacyl-CoA to acyl-CoA | Cupriavidus necator |
2.3.1.9 | additional information | the enzyme functions as a dimer, and the monomer comprises three subdomains I, II, and III. The structural comparison between the apoform and the CoA-bound form reveals that the enzyme undergoes a structural change in the lid-subdomain III upon the binding of the CoA substrate. The CoA molecule is stabilized by hydrogen bonding with positively charged residues Lys18, Arg210, and Arg217, and residues Thr213 and Gln151 aid its binding as well. At the enzyme's active site highly conserved residues, Cys91, His348, and Cys378, are located near the thiol-group of CoA, indicating that enzyme ReH16_A1887 might catalyze the thiolase reaction in a way similar to other thiolases. In the vicinity of the covalent nucleophile Cys91, a hydrophobic hole that might serve as a binding site for the acyl-group of 3-oxoacyl-CoA. Subdomains I and II harbor the active site residues: Cys91 in subdomain I, and His348 and Cys378 in subdomain II | Cupriavidus necator |