Literature summary for 2.3.1.16 extracted from

Kiema, T.R.; Harijan, R.K.; Strozyk, M.; Fukao, T.; Alexson, S.E.; Wierenga, R.K.
The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities (2014), Acta Crystallogr. Sect. D, 70, 3212-3225.

Cloned(Commentary)

Cloned (Comment)	Organism
gene ACAA2, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and mutant enzymes in apoform and in complex with CoA, hanging drop vapour diffusion method, mixing of 0.002 ml of 4.4 mg/ml protein in 25 mM Tris-HCl pH 8.0, 1 mM DTT, with 0.002 ml of reservoir solution containing 100 mM MES, pH 6.6, or MOPS, pH 7.2, and 14-15% PEG MME 5000, equilibration against 1 ml of reservoir solution, 22°C, method optimization, X-ray diffraction structure determination and analysis at 2.0-3.3 A resolution, modeling	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant wild-type and mutant enzymes in apoform and in complex with CoA, hanging drop vapour diffusion method, mixing of 0.002 ml of 4.4 mg/ml protein in 25 mM Tris-HCl pH 8.0, 1 mM DTT, with 0.002 ml of reservoir solution containing 100 mM MES, pH 6.6, or MOPS, pH 7.2, and 14-15% PEG MME 5000, equilibration against 1 ml of reservoir solution, 22°C, method optimization, X-ray diffraction structure determination and analysis at 2.0-3.3 A resolution, modeling

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
C382A	site-directed mutagenesis	Homo sapiens
C92A	site-directed mutagenesis	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0092	-	acetoacetyl-CoA	pH 7.0, 25°C, recombinant wild-type enzyme, degradative reaction	Homo sapiens
0.25	-	acetyl-CoA	pH 7.0, 25°C, recombinant wild-type enzyme, synthesis reaction	Homo sapiens

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
mitochondrion	-	Homo sapiens	5739	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
2 acetyl-CoA	Homo sapiens	reaction of EC 2.3.1.9	CoA + acetoacetyl-CoA	-	r
acyl-CoA + acetyl-CoA	Homo sapiens	-	CoA + 3-oxoacyl-CoA	-	?
CoA + acetoacetyl-CoA	Homo sapiens	-	2 acetyl-CoA	-	r
additional information	Homo sapiens	the enzyme catalyzes the thiolytic cleavage of medium-chain to long-chain 3-oxoacyl-CoAs to acetyl-CoA and a fatty acyl-CoA shortened by two C atom, the reaction involves residues C382, C92, and H352, overview. The enzyme can also catalyze the condensation of two acetyl-CoA molecules into acetoacetyl-CoA, EC 2.3.1.9	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P42765	gene ACAA2; gene ACAA2	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, cation exchange chromatography, ultrafiltration, and gel filtration, tag cleavage by thrombin	Homo sapiens

Reaction

Reaction	Comment	Organism	Reaction ID
acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA	mechanism of the reverse, degradative thiolase reaction, overview. The mode of binding of the acyl moiety favours its transfer to CoA compared with water. Both the thiolytic cleavage and the condensation reaction are catalysed via covalent modification of the nucleophilic cysteine (Cys92) and by stabilization of the negatively charged reaction intermediates by oxyanion holes. The nucleophilic cysteine attacks the beta-carbon of the 3-ketoacyl-CoA and becomes covalently modified. Another cysteine, Cys382, acts first as an acid providing a proton for the leaving acetyl-CoA and next as a base abstracting a proton from the incoming CoA. The activated CoA molecule then attacks the carbonyl C atom of the acylated cysteine and the fatty acyl-CoA is released. The cysteine acting as the nucleophile is activated by a conserved histidine residue (His352). This protonated histidine subsequently forms OAH1 together with a water molecule which is hydrogen-bonded to Asn320. The rate-limiting steps are different in the degradative and biosynthetic thiolases	Homo sapiens	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
adrenal gland	-	Homo sapiens	-
heart	-	Homo sapiens	-
kidney	-	Homo sapiens	-
liver	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 acetyl-CoA	reaction of EC 2.3.1.9	Homo sapiens	CoA + acetoacetyl-CoA	-	r
3-oxodecanoyl-CoA + CoA	degradation of 3-oxodecanoyl-CoA into acetyl-CoA and octanoyl-CoA by human mitochondrial 3-ketoacyl-CoA thiolase, substrate binding mode and reaction mechanism, overview	Homo sapiens	acetyl-CoA + octanoyl-CoA	-	?
acyl-CoA + acetyl-CoA	-	Homo sapiens	CoA + 3-oxoacyl-CoA	-	?
CoA + acetoacetyl-CoA	-	Homo sapiens	2 acetyl-CoA	-	r
additional information	the enzyme catalyzes the thiolytic cleavage of medium-chain to long-chain 3-oxoacyl-CoAs to acetyl-CoA and a fatty acyl-CoA shortened by two C atom, the reaction involves residues C382, C92, and H352, overview. The enzyme can also catalyze the condensation of two acetyl-CoA molecules into acetoacetyl-CoA, EC 2.3.1.9	Homo sapiens	?	-	?
additional information	active site structure and CoA binding structure, and enzyme substrate specificity, detailed overview	Homo sapiens	?	-	?

Subunits

Subunits	Comment	Organism
homotetramer	thiolases share a structurally conserved thiolase core domain composed of topologically similar N-terminal and C-terminal subdomains and a more variable loop domain with structural features involved in the tetramerization and substrate specificity	Homo sapiens

Synonyms

Synonyms	Comment	Organism
mitochondrial 3-ketoacyl-CoA thiolase	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
1.4	-	acetyl-CoA	pH 7.0, 25°C, recombinant wild-type enzyme, synthesis reaction	Homo sapiens
14.8	-	acetoacetyl-CoA	pH 7.0, 25°C, recombinant wild-type enzyme, degradative reaction	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	thiolase reaction	Homo sapiens

General Information

General Information	Comment	Organism
evolution	the rate-limiting steps are different in the degradative and biosynthetic thiolases. Thiolases share a structurally conserved thiolase core domain composed of topologically similar N-terminal and C-terminal subdomains and a more variable loop domain with structural features involved in the tetramerization and substrate specificity	Homo sapiens
additional information	the nucleophilic cysteine attacks the beta-carbon of the 3-ketoacyl-CoA and becomes covalently modified. Another cysteine, Cys382, acts first as an acid providing a proton for the leaving acetyl-CoA and next as a base abstracting a proton from the incoming CoA. The activated CoA molecule then attacks the carbonyl C atom of the acylated cysteine and the fatty acyl-CoA is released. The cysteine acting as the nucleophile is activated by a conserved histidine residue (His352)	Homo sapiens
physiological function	thiolases are enzymes which are involved in lipid metabolism, catalysing the biological Claisen condensation and thiolytic cleavage reactions using a cysteine thiol in the active site. The enzyme catalyzes the last step of the mitochondrial beta-oxidation pathway and is also involved in the synthesis of acetoacetyl-CoA for the generation of ketone bodies, cf. EC 2.3.1.9	Homo sapiens