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Literature summary for 2.3.1.9 extracted from

  • Kim, J.; Kim, K.J.
    Crystal structure and biochemical properties of ReH16_A1887, the 3-ketoacyl-CoA thiolase from Ralstonia eutropha H16 (2015), Biochem. Biophys. Res. Commun., 459, 547-552.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
recombinant overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Cupriavidus necator

Crystallization (Commentary)

Crystallization (Comment) Organism
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

Protein Variants

Protein Variants Comment Organism
C378A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
C91A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
H348A site-directed mutagenesis, almost inactive mutant Cupriavidus necator
K18A site-directed mutagenesis, the mutant shows over 80% reduced activity compared to the wild-type enzyme Cupriavidus necator
K217A site-directed mutagenesis, the mutant shows 60% reduced activity compared to the wild-type enzyme Cupriavidus necator
Q151A site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme Cupriavidus necator
R210A site-directed mutagenesis, the mutant shows 70% reduced activity compared to the wild-type enzyme Cupriavidus necator

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Cupriavidus necator Q0KAI3 gene H16_A1887; gene H16_A1887
-
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 Q0KAI3 gene H16_A1887; gene H16_A1887
-

Purification (Commentary)

Purification (Comment) Organism
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 acetyl-CoA
-
Cupriavidus necator CoA + acetoacetyl-CoA
-
r
2 acetyl-CoA
-
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 CoA + acetoacetyl-CoA
-
r
CoA + acetoacetyl-CoA
-
Cupriavidus necator 2 acetyl-CoA
-
r
CoA + acetoacetyl-CoA CoA binding mode, overview Cupriavidus necator 2 acetyl-CoA
-
r
CoA + acetoacetyl-CoA
-
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 2 acetyl-CoA
-
r
CoA + acetoacetyl-CoA CoA binding mode, overview Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1 2 acetyl-CoA
-
r
additional information the enzyme shows degradative thiolase activity by converting 3-oxoacyl-CoA to acyl-CoA Cupriavidus necator ?
-
?
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 ?
-
?

Subunits

Subunits Comment Organism
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

Synonyms

Synonyms Comment Organism
3-ketoacyl-CoA thiolase
-
Cupriavidus necator
A1887
-
Cupriavidus necator
ACAT
-
Cupriavidus necator
acetoacetyl-CoA thiolase
-
Cupriavidus necator
KACT
-
Cupriavidus necator
type II thiolase
-
Cupriavidus necator

General Information

General Information Comment Organism
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
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