# Thiolase
Thiolases are coenzyme A (CoA)-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA into an acyl-CoA shortened by two carbons plus acetyl-CoA, and the reverse reaction, a thioester-dependent [Claisen condensation](https://www.edgepedia.org/claisen-condensation) that builds carbon-carbon bonds.[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) They occur throughout prokaryotes and eukaryotes and sit at central points of metabolism: the final step of each cycle of fatty acid β-oxidation, the first committed step of ketone body and sterol biosynthesis, and the production of acetoacetyl-CoA in solvent-producing bacteria.[2](https://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Oxford-Dictionary-of-Biochemistry-and-Molecular-Biology-by-Teresa-K--Attwood-et--al---2006-.pdf) • [3](https://pmc.ncbi.nlm.nih.gov/articles/PMC3026612/)
| Key fact | Detail |
|---|---|
| Reaction | Reversible thiolytic cleavage of 3-ketoacyl-CoA to acyl-CoA and acetyl-CoA; reverse direction is the thioester-dependent Claisen condensation[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) |
| Enzyme classes | Thiolase I (EC 2.3.1.16, degradative 3-ketoacyl-CoA thiolase) and thiolase II (EC 2.3.1.9, acetoacetyl-CoA thiolase)[4](https://pubmed.ncbi.nlm.nih.gov/15980957/) |
| Catalytic machinery | Two reactive cysteines (nucleophile and acid/base), two oxyanion holes, two active site waters, four catalytic loops[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) |
| Quaternary structure | Tight dimers or tetramers (dimers of dimers)[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) |
| Human isoenzymes | Six thiolases (CT, T1, T2, SCP2, AB, TFE) in cytosol, mitochondria, and peroxisomes[11](https://journals.asm.org/doi/10.1128/aem.02372-21) |
| Substrate specificity | Thiolase I acts on substrates of 4 to 22 carbons; thiolase II is specific for C4 chains[11](https://journals.asm.org/doi/10.1128/aem.02372-21) |
| Related disease | Beta-ketothiolase (T2) deficiency, an autosomal recessive inborn error of isoleucine and ketone body metabolism caused by ACAT1 variants[5](https://pubmed.ncbi.nlm.nih.gov/30393371/) |
## The reaction and its mechanism
The degradative (thiolysis) reaction cleaves a 3-ketoacyl-CoA: the enzyme's nucleophilic cysteine attacks the thioester, forming a covalent acyl-enzyme intermediate and releasing acetyl-CoA, after which free CoA liberates the shortened acyl-CoA.[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) • [6](https://www.ncbi.nlm.nih.gov/Structure/cdd/cd00751) In the biosynthetic direction, two acetyl-CoA molecules condense to acetoacetyl-CoA in a biological Claisen condensation, a reaction that extends an acyl-CoA chain by a two-carbon acetyl module.[3](https://pmc.ncbi.nlm.nih.gov/articles/PMC3026612/) The condensation is thermodynamically unfavorable, which matters in bacteria such as *Clostridium acetobutylicum*, where thiolase competes with other acetyl-CoA-consuming enzymes and is inhibited in the condensation direction by micromolar levels of CoA.[7](https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/thiolase) • [8](https://pubmed.ncbi.nlm.nih.gov/16347774/)
**Two cysteines and two oxyanion holes.** All thiolases have two reactive cysteines: a nucleophilic cysteine that forms the covalent intermediate and an acid/base cysteine.[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) In the best-characterized enzyme, the biosynthetic thiolase of *Zoogloea ramigera*, the nucleophile is Cys89 and the base is Cys378; Cys89, Asn316, and His348 form the CNH catalytic triad of the thiolase superfamily.[9](https://pubmed.ncbi.nlm.nih.gov/19842716/) Oxyanion hole I stabilizes the enolate intermediate generated from acetyl-CoA during condensation, while oxyanion hole II stabilizes the tetrahedral intermediate of the acetylated enzyme.[9](https://pubmed.ncbi.nlm.nih.gov/19842716/) Mutational studies show the asymmetry of the two directions: replacing Asn316 with aspartate abolishes detectable activity, and removing either hydrogen bond donor of oxyanion hole I costs at least 3.4 kcal/mol of transition state stabilization for condensation, while thiolytic degradation is much less affected because oxyanion hole I is not involved in stabilizing the transition state of its rate-limiting step.[9](https://pubmed.ncbi.nlm.nih.gov/19842716/) His348 has a dual role, contributing to oxyanion hole I and activating Cys89.[9](https://pubmed.ncbi.nlm.nih.gov/19842716/)
The active site also contains two conserved water molecules, Wat82 and Wat49, which help form oxyanion hole I and influence the reactivity of the catalytic base Cys378, respectively.[9](https://pubmed.ncbi.nlm.nih.gov/19842716/) In terms of structure, two oxyanion holes, a pair of active site waters, and four catalytic loops bearing distinctive amino acid sequence fingerprints delineate the active site; the three thiolase subfamilies are distinguishable because each one's catalytic loop carries its own unique fingerprint, giving rise to distinct active site properties.[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS) In the yeast peroxisomal thiolase, the catalytic triad of Cys125, His375, and Cys403 sits at the bottom of a cylindrical pocket comprising the active site.[10](https://academic.oup.com/femsre/article/27/1/35/500066)
## Types, locations, and evolution
Thiolases fall into two functional classes. Thiolase I (EC 2.3.1.16) catalyzes the thiolytic cleavage of oxidized acyl-CoA in β-oxidation and effectively catalyzes only the degradative reaction, which is thermodynamically favorable; thiolase II (EC 2.3.1.9) synthesizes or degrades acetoacetyl-CoA and can also act in the last thiolytic cleavage of the fatty acid β-oxidation spiral.[4](https://pubmed.ncbi.nlm.nih.gov/15980957/) • [11](https://journals.asm.org/doi/10.1128/aem.02372-21) The two classes share significant sequence similarity and essentially the same active site residues, indicating a common ancestor.[11](https://journals.asm.org/doi/10.1128/aem.02372-21)
In eukaryotes, thiolases are localized in three compartments: the peroxisome, the mitochondrion, and the cytosol.[4](https://pubmed.ncbi.nlm.nih.gov/15980957/) The human genome encodes six thiolases, named CT, T1, T2, SCP2, AB, and TFE, differing in sequence, oligomeric state, substrate specificity, and localization: only CT belongs to thiolase II and is cytosolic, SCP2 and AB are peroxisomal degradative enzymes, and T1, T2, and TFE are mitochondrial degradative enzymes.[11](https://journals.asm.org/doi/10.1128/aem.02372-21) Some eukaryotes carry an additional peroxisomal thiolase I called SCP-X, specific for long- and branched-chain fatty acids, sometimes fused with a sterol carrier protein.[11](https://journals.asm.org/doi/10.1128/aem.02372-21) In fungi such as *Aspergillus oryzae*, six thiolase genes were identified with distinct roles in fatty acid and ergosterol biosynthesis.[11](https://journals.asm.org/doi/10.1128/aem.02372-21)
Phylogenetic analysis of eukaryotic thiolase sequences shows six distinct clusters that correlate with class and subcellular location, all emerging close to proteobacterial sequences; the analysis suggests eukaryotic peroxisomal and mitochondrial thiolases may have been acquired from delta-proteobacteria before the ancestor of all known eukaryotes.[4](https://pubmed.ncbi.nlm.nih.gov/15980957/)
## Roles in metabolism
**Fatty acid β-oxidation.** Thiolytic cleavage by 3-ketoacyl-CoA thiolase is the concluding reaction of each cycle of β-oxidation, shortening the acyl-CoA by two carbons and producing one acetyl-CoA per round.[2](https://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Oxford-Dictionary-of-Biochemistry-and-Molecular-Biology-by-Teresa-K--Attwood-et--al---2006-.pdf) Mitochondrial and peroxisomal β-oxidation systems are chemically identical in this thiolytic step but use different enzymes encoded by distinct genes.[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729952/) In plant peroxisomes, the 3-ketoacyl-CoA thiolase's structure and activity are regulated by a sensitive redox switch.[1](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS)
**Ketone body and sterol metabolism.** In ketogenesis, thiolase condenses two acetyl-CoA into acetoacetyl-CoA, the precursor of the ketone bodies; during ketone body utilization in mitochondria, acetoacetyl-CoA is cleaved by thiolase to yield two acetyl-CoA molecules for the TCA cycle.[13](https://discovery.ucl.ac.uk/id/eprint/10053453/1/Khabbush_10053453_thesis.Redacted.pdf) Cytosolic thiolase II likewise supplies acetoacetyl-CoA for sterol biosynthesis.[11](https://journals.asm.org/doi/10.1128/aem.02372-21)
**Bacterial solvent production.** In *C. acetobutylicum*, thiolase (acetyl-CoA acetyltransferase, EC 2.3.1.9) performs the thermodynamically unfavorable condensation of two acetyl-CoA to acetoacetyl-CoA, the precursor of the four-carbon products of the fermentation. Kinetic analysis of the thiolysis reaction indicated a ping-pong binding mechanism with Km values of 4.7 and 32 μmol for CoA and acetoacetyl-CoA, respectively.[7](https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/thiolase)
## Beta-ketothiolase deficiency
Deficiency of mitochondrial acetoacetyl-CoA thiolase (T2, encoded by the [ACAT1](https://www.edgepedia.org/acat1) gene), also called beta-ketothiolase deficiency (OMIM #203750), is an autosomal recessive inborn error of metabolism affecting isoleucine catabolism and ketone body metabolism.[5](https://pubmed.ncbi.nlm.nih.gov/30393371/) • [14](https://pmc.ncbi.nlm.nih.gov/articles/PMC7187484/) It is clinically characterized by intermittent ketoacidotic crises under ketogenic stresses, which can induce severe, potentially life-threatening metabolic acidosis.[5](https://pubmed.ncbi.nlm.nih.gov/30393371/) • [15](https://www.sciencedirect.com/science/article/pii/S1096719226005251) In reported patient series, most affected individuals developed their first ketoacidotic crisis between the ages of 6 months and 3 years, and most experienced fewer than three metabolic crises.[5](https://pubmed.ncbi.nlm.nih.gov/30393371/) Neurological manifestations, especially extrapyramidal ones, can occur as sequelae of severe metabolic acidosis, and can also appear in patients without any apparent metabolic crisis.[5](https://pubmed.ncbi.nlm.nih.gov/30393371/) A 2019 mutation update compiled 105 disease-associated ACAT1 variants reported in 149 patients from 134 families; structural analysis suggests most missense variants concern residues buried in the mature protein, predicted to impair stability or folding and thereby reduce the capacity to degrade 2-methylacetoacetyl-CoA and acetoacetyl-CoA.[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC6790690/) [Newborn screening](https://www.edgepedia.org/newborn-screening) is performed in some countries, but some affected patients have been missed, so T2 deficiency should be considered in patients with severe metabolic acidosis even where screening exists.[5](https://pubmed.ncbi.nlm.nih.gov/30393371/)
## References
1. [Harijan RK, Dalwani S, Kiema T-R, Venkatesan R, Wierenga RK (2023). Thiolase: A Versatile Biocatalyst Employing Coenzyme A–Thioester Chemistry for Making and Breaking C–C Bonds. Annual Review of Biochemistry 92:351–384.](https://www.annualreviews.org/content/journals/10.1146/annurev-biochem-052521-033746?TRACK=RSS)
2. [acetyl-CoA C-acyltransferase, EC 2.3.1.16, Oxford Dictionary of Biochemistry and Molecular Biology (2006).](https://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Oxford-Dictionary-of-Biochemistry-and-Molecular-Biology-by-Teresa-K--Attwood-et--al---2006-.pdf)
3. [Enzymes of the Mevalonate Pathway of Isoprenoid Biosynthesis, PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC3026612/)
4. [Peretó J, López-García P, Moreira D (2005). Phylogenetic analysis of eukaryotic thiolases suggests multiple proteobacterial origins. J Mol Evol 61:65–74.](https://pubmed.ncbi.nlm.nih.gov/15980957/)
5. [Recent advances in understanding beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency. Journal of Human Genetics (2018).](https://pubmed.ncbi.nlm.nih.gov/30393371/)
6. [CDD Conserved Protein Domain Family: thiolase, NCBI.](https://www.ncbi.nlm.nih.gov/Structure/cdd/cd00751)
7. [Thiolase, ScienceDirect Topics overview (Biotechnology Advances, 2009).](https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/thiolase)
8. [Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents, PubMed.](https://pubmed.ncbi.nlm.nih.gov/16347774/)
9. [Meriläinen G, Poikela V, Kursula P, Wierenga RK (2009). The thiolase reaction mechanism: the importance of Asn316 and His348 for stabilizing the enolate intermediate of the Claisen condensation. Biochemistry 48:11011–25.](https://pubmed.ncbi.nlm.nih.gov/19842716/)
10. [Hiltunen JK et al. (2003). The biochemistry of peroxisomal β-oxidation in the yeast Saccharomyces cerevisiae. FEMS Microbiology Reviews 27:35–64.](https://academic.oup.com/femsre/article/27/1/35/500066)
11. [Identification of Six Thiolases and Their Effects on Fatty Acid and Ergosterol Biosynthesis in Aspergillus oryzae. Applied and Environmental Microbiology (2021).](https://journals.asm.org/doi/10.1128/aem.02372-21)
12. [Metabolic Interplay between Peroxisomes and Other Subcellular Organelles, PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC4729952/)
13. [Khabbush A (2017). Thesis, University College London Discovery repository.](https://discovery.ucl.ac.uk/id/eprint/10053453/1/Khabbush_10053453_thesis.Redacted.pdf)
14. [2-methylacetoacetyl-coenzyme A thiolase (beta-ketothiolase) deficiency: one disease - two pathways, PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC7187484/)
15. [Beta-ketothiolase deficiency: two novel ACAT1 variants and a retrospective study of 76 cases in China. Molecular Genetics and Metabolism.](https://www.sciencedirect.com/science/article/pii/S1096719226005251)
16. [Abdelkreem E et al. (2019). Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, PMC.](https://pmc.ncbi.nlm.nih.gov/articles/PMC6790690/)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Metabolism and metabolic pathways › Lipid and fatty acid metabolism › Lipid metabolism enzyme families and activities › Beta-oxidation and acyl-CoA handling enzymes*
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