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Literature summary extracted from

  • Kim, J.; Kim, K.J.
    Crystal structure and biochemical characterization of a 3-ketoacyl-CoA thiolase from Ralstoniaeutropha H16 (2016), Int. J. Biol. Macromol., 82, 425-431.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.9 gene ReH16_B0759, DNA and amino acid sequence determination and analysis, recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Cupriavidus necator

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.1.9 purified recombinant enzyme in apoform and with bound CoA, hanging drop vapor diffusion method, mixing 0.001 ml of 40 mg/ml protein in 40 mM Tris-HCl, pH 8.0, and 5 mM 2-mercaptoethanol, with 0.001 ml of reservoir solution containing 1.0 M ammonium sulfate, 0.1 M HEPES, pH 7.25, and equilibration against 0.5 ml of reservoir solution, 20°C, 3 days, X-ray diffraction structure determination and analysis at 2.0-2.3 A resolution, molecular replacement method using the structure of Mycobacterium tuberculosis thiolase Mttt0182, PDB ID1ULQ, as a search model, structure modeling Cupriavidus necator

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.9 C377A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
2.3.1.9 C89A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
2.3.1.9 F156A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Cupriavidus necator
2.3.1.9 H347A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
2.3.1.9 K17A site-directed mutagenesis, the mutant shows 60% reduced activity compared to the wild-type enzyme Cupriavidus necator
2.3.1.9 M124A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Cupriavidus necator
2.3.1.9 R220A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Cupriavidus necator
2.3.1.9 V231A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Cupriavidus necator

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.9 additional information
-
 additional information Michaelis-Menten kinetics Cupriavidus necator

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.9 CoA + acetoacetyl-CoA Cupriavidus necator
-
 2 acetyl-CoA
-
 ?
2.3.1.9 CoA + acetoacetyl-CoA Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
-
 2 acetyl-CoA
-
 ?
2.3.1.9 additional information Cupriavidus necator the enzyme shows degradative thiolase activity by converting 3-ketoacyl-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-ketoacyl-CoA to acyl-CoA ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.9 Cupriavidus necator Q0K368 gene H16_B0759; gene H16_B0759
-
2.3.1.9 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 Q0K368 gene H16_B0759; gene H16_B0759
-

Purification (Commentary)

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.9 CoA + acetoacetyl-CoA
-
 Cupriavidus necator 2 acetyl-CoA
-
 ?
2.3.1.9 CoA + acetoacetyl-CoA CoA substrate binding structure, overview Cupriavidus necator 2 acetyl-CoA
-
 ?
2.3.1.9 CoA + acetoacetyl-CoA
-
 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 2 acetyl-CoA
-
 ?
2.3.1.9 CoA + acetoacetyl-CoA CoA substrate binding structure, overview Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 2 acetyl-CoA
-
 ?
2.3.1.9 additional information the enzyme shows degradative thiolase activity by converting 3-ketoacyl-CoA to acyl-CoA Cupriavidus necator ?
-
 ?
2.3.1.9 additional information the bound CoA substrate is located within the deep cleft formed by subdomains I, II, and III. The thiol group of CoA is located in the vicinity of the catalytic site for the degradation reaction, whereas,the adenosine nucleotide moiety is exposed on the surface. Unlike enzyme ReH16_A1887, that shows substantial structural changeupon the binding of CoA, the CoA-bound form of enzyme ReH16_B0759 exhibits quite similar structure to the apoform of the protein except for residual movement of Arg220. Moreover, the CoA binding mode of enzyme ReH16_B0759 is quite different from that of enzyme ReH16_A1887 Cupriavidus necator ?
-
 ?
2.3.1.9 additional information the enzyme shows degradative thiolase activity by converting 3-ketoacyl-CoA to acyl-CoA Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 ?
-
 ?
2.3.1.9 additional information the bound CoA substrate is located within the deep cleft formed by subdomains I, II, and III. The thiol group of CoA is located in the vicinity of the catalytic site for the degradation reaction, whereas,the adenosine nucleotide moiety is exposed on the surface. Unlike enzyme ReH16_A1887, that shows substantial structural changeupon the binding of CoA, the CoA-bound form of enzyme ReH16_B0759 exhibits quite similar structure to the apoform of the protein except for residual movement of Arg220. Moreover, the CoA binding mode of enzyme ReH16_B0759 is quite different from that of enzyme ReH16_A1887 Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.3.1.9 3-ketoacyl-CoA thiolase
-
 Cupriavidus necator
2.3.1.9 3-ketoacyl-coenzyme A thiolase
-
 Cupriavidus necator
2.3.1.9 ACAT
-
 Cupriavidus necator
2.3.1.9 KACT
-
 Cupriavidus necator
2.3.1.9 ReH16_B0759
-
 Cupriavidus necator
2.3.1.9 type II thiolase
-
 Cupriavidus necator

General Information

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-ketoacyl-CoA to acyl-CoA Cupriavidus necator
2.3.1.9 additional information similar to other degradative thiolases, enzyme ReH16_B0759 functions as a dimer, and the monomer comprises three subdomains. Unlike enzyme ReH16_A1887, a substantial structural change is not observed upon the binding of the CoA substrate in enzyme ReH16_B0759. At the active site of the enzyme highly conserved residues Cys89, His347, and Cys377are located near the thiol-group of CoA Cupriavidus necator