GO:0003988 acetyl-CoA C-acyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0003988 acetyl-CoA C-acyltransferase activity catalyzes the reversible reaction acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA, a central step in fatty acid beta-oxidation and ketone body metabolism.
• The term is a molecular_function in the Gene Ontology and is commonly known as 3-ketoacyl-CoA thiolase or beta-ketothiolase activity.
• Key enzymes carrying this activity include ACAA2, ACAT1, and the mitochondrial trifunctional protein subunit HADHA/HADHB, which are essential for energy production.
• Defects in this activity are linked to metabolic disorders such as LCHADD-associated chorioretinopathy and hepatic lipid dysregulation.
• Studying this activity requires integrating genetic models (knockout, point mutation, knock-in, overexpression) with biochemical and proteomic methods.
• CRISPR-based cell models are powerful tools to dissect the role of acetyl-CoA C-acyltransferase activity in metabolism and disease.
Description
Acetyl-CoA C-acyltransferase activity (GO:0003988) is a fundamental enzymatic activity that catalyzes the cleavage of 3-ketoacyl-CoA into acetyl-CoA and a shortened acyl-CoA, or the reverse condensation reaction. This activity is essential for fatty acid beta-oxidation, ketone body metabolism, and the final steps of mitochondrial fatty acid oxidation. Researchers study this activity to understand energy homeostasis, metabolic disorders, and potential therapeutic targets in cancer and metabolic diseases. The enzyme is also known as 3-ketoacyl-CoA thiolase, beta-ketothiolase, or acetoacetyl-CoA beta-ketothiolase, reflecting its broad substrate specificity and reversible nature. In this article, we provide a comprehensive overview of GO:0003988, covering its definition, mechanism, key genes, disease associations, and modern research methods including CRISPR-based models.
acetyl-CoA C-acyltransferase activity At A Glance
| GO ID | GO:0003988 |
|---|---|
| GO term | acetyl-CoA C-acyltransferase activity |
| Ontology | molecular_function |
| Synonym | 3-ketoacyl-CoA thiolase activity; beta-ketothiolase activity; acetoacetyl-CoA beta-ketothiolase activity; acetyl-CoA acyltransferase activity |
| Major function | Catalyzes the reversible transfer of an acetyl group from acetyl-CoA to acyl-CoA, producing CoA and 3-oxoacyl-CoA |
| Reaction | acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA |
| Related pathways | Fatty acid beta-oxidation, ketone body metabolism, mitochondrial energy production |
| Key enzymes | ACAA2, ACAT1, HADHA, HADHB, and other thiolases |
What Is GO:0003988?
According to the Gene Ontology, acetyl-CoA C-acyltransferase activity (GO:0003988) is defined as the catalysis of the reaction: acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA. In other words, it transfers an acetyl group from acetyl-CoA to an acyl-CoA acceptor, forming a 3-oxoacyl-CoA and free CoA. This activity is reversible and is central to the thiolytic cleavage of 3-ketoacyl-CoA during fatty acid beta-oxidation, as well as to the synthesis of ketone bodies and other metabolic intermediates.
Why Is acetyl-CoA C-acyltransferase activity Important in Cell Biology?
Acetyl-CoA C-acyltransferase activity is critical for cellular energy metabolism because it catalyzes the final step of fatty acid beta-oxidation, generating acetyl-CoA that enters the tricarboxylic acid cycle. It also plays a key role in ketone body metabolism, which is vital during fasting and for extrahepatic tissues such as muscle and brain. Dysregulation of this activity has been implicated in metabolic disorders, including hepatic lipid dysregulation and LCHADD-associated chorioretinopathy. Furthermore, recent studies highlight its importance in antitumor immunity and muscle ketolysis, making it a promising target for therapeutic intervention.
• Essential for fatty acid beta-oxidation and energy production in mitochondria.
• Central to ketone body metabolism, supporting extrahepatic tissues during fasting.
• Mutations or deficiencies lead to metabolic diseases such as LCHADD.
• Modulates hepatic lipid dynamics and lipid accumulation.
• Involved in antitumor immunity through mitochondrial trifunctional protein.
• Regulates muscle ketolysis and exercise capacity.
• Target of drug-induced liver injury, e.g., by Diosbulbin B.
• Potential biomarker for myocardial ischemia/reperfusion injury.
• Plays a role in acetate generation in liver mitochondria.
• Provides a model for studying enzyme kinetics and substrate specificity.
Molecular Mechanism of acetyl-CoA C-acyltransferase activity
Substrate Binding and Catalytic Mechanism
In simple terms: The enzyme grabs an acyl-CoA molecule and an acetyl-CoA, then swaps parts to make a new 3-oxoacyl-CoA and free CoA.
The catalytic mechanism of acetyl-CoA C-acyltransferase involves a ping-pong bi-bi kinetic mechanism where an acetyl group is first transferred from acetyl-CoA to a cysteine residue in the active site, forming an acetyl-enzyme intermediate. The acyl-CoA substrate then enters, and the acetyl group is transferred to its beta-carbon, yielding a 3-oxoacyl-CoA and releasing CoA. This reversible reaction is essential for both beta-oxidation and ketogenesis.
Role in Fatty Acid Beta-Oxidation
In simple terms: This enzyme performs the last cut in the process that breaks down fatty acids to produce energy.
In mitochondrial fatty acid beta-oxidation, acetyl-CoA C-acyltransferase catalyzes the thiolytic cleavage of 3-ketoacyl-CoA, releasing acetyl-CoA and a shortened acyl-CoA that re-enters the cycle. This activity is carried out by several enzymes, including ACAA2 and the trifunctional protein (HADHA/HADHB), which are essential for energy production from fats.
Ketone Body Metabolism
In simple terms: The enzyme helps produce ketone bodies, which are alternative fuels used during fasting or exercise.
In ketogenesis, acetyl-CoA C-acyltransferase activity catalyzes the condensation of two acetyl-CoA molecules to form acetoacetyl-CoA, a precursor of ketone bodies. This activity is particularly important in the liver and in extrahepatic tissues such as muscle, where ketone bodies are oxidized for energy. The muscle-specific MEF2Dα2 isoform promotes ketolysis by regulating this activity.
Structural Components and Assembly
In simple terms: The enzyme is usually a multi-subunit complex, and its structure determines what substrates it can handle.
Acetyl-CoA C-acyltransferase enzymes typically function as homodimers or as part of larger multienzyme complexes such as the mitochondrial trifunctional protein (MTP), which consists of HADHA and HADHB subunits. The active site contains a catalytic cysteine residue that forms a covalent acetyl-enzyme intermediate. The assembly of these complexes is regulated by cellular energy status and substrate availability.
Regulation and Cofactors
In simple terms: The enzyme's activity can be turned up or down by other molecules and by the cell's energy needs.
Acetyl-CoA C-acyltransferase activity is regulated by substrate availability, product inhibition, and post-translational modifications. For example, spermidine activates the mitochondrial trifunctional protein, enhancing antitumor immunity. In chicken embryos, ACAA2 modulates hepatic lipid dynamics, suggesting nutritional and hormonal regulation. No specific cofactors are required beyond CoA and acyl-CoA substrates.
Key Genes Involved in GO:0003988 acetyl-CoA C-acyltransferase activity
The following genes encode enzymes that exhibit acetyl-CoA C-acyltransferase activity or are directly involved in its regulation and function.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACAA2 | Mitochondrial 3-ketoacyl-CoA thiolase; catalyzes final step of beta-oxidation | Target for hepatic lipid dysregulation and metabolic disorders |
| ACAT1 | Acetoacetyl-CoA thiolase; involved in ketone body metabolism and isoleucine degradation | Studied in cancer metabolism and ketogenesis |
| HADHA | Subunit of mitochondrial trifunctional protein; carries long-chain enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities | Linked to LCHADD and antitumor immunity |
| HADHB | Subunit of mitochondrial trifunctional protein; carries 3-ketoacyl-CoA thiolase activity | Mutations cause LCHADD and neuropathy |
| ACAA1 | Peroxisomal 3-ketoacyl-CoA thiolase; involved in very-long-chain fatty acid oxidation | Studied in peroxisomal disorders |
| ACAT2 | Cytosolic acetoacetyl-CoA thiolase; involved in cholesterol synthesis | Target of Diosbulbin B-induced liver injury |
| MEF2D | Transcription factor regulating muscle ketolysis and MEF2Dα2 isoform | Promotes running capacity and ketolysis in muscle |
| PDHA1 | Pyruvate dehydrogenase subunit; links glycolysis to acetyl-CoA production | Affected in myocardial ischemia/reperfusion injury |
| CPT1A | Carnitine palmitoyltransferase 1A; rate-limiting for fatty acid entry into mitochondria | Upstream of beta-oxidation and thiolase activity |
| CPT2 | Carnitine palmitoyltransferase 2; involved in fatty acid oxidation | Defects cause metabolic myopathies |
| SLC25A20 | Carnitine-acylcarnitine translocase; transports acylcarnitines into mitochondria | Linked to fatty acid oxidation disorders |
| ETFA | Electron transfer flavoprotein alpha subunit; supports beta-oxidation | Associated with glutaric acidemia type II |
| ETFB | Electron transfer flavoprotein beta subunit; supports beta-oxidation | Associated with glutaric acidemia type II |
| ETFDH | Electron transfer flavoprotein dehydrogenase; supports beta-oxidation | Associated with glutaric acidemia type II |
| HMGCS2 | Mitochondrial HMG-CoA synthase; ketogenesis | Regulated by ketogenic signals |
| BDH1 | 3-hydroxybutyrate dehydrogenase; ketone body utilization | Studied in ketone metabolism |
| OXCT1 | Succinyl-CoA:3-ketoacid CoA transferase; ketolysis | Muscle ketolysis and exercise capacity |
| ACAT1 | Acetyl-CoA acetyltransferase 1; ketogenesis and isoleucine degradation | Target for cancer metabolism |
How Is acetyl-CoA C-acyltransferase activity Regulated?
Acetyl-CoA C-acyltransferase activity is regulated at multiple levels. Substrate availability, particularly of acyl-CoA and acetyl-CoA, directly influences flux through the reaction. Hormonal signals such as insulin and glucagon modulate the expression of genes encoding these enzymes, especially during fasting and feeding. Post-translational modifications, including phosphorylation and acetylation, can alter enzyme activity. In muscle, the MEF2Dα2 isoform promotes ketolysis by upregulating genes involved in ketone body utilization, including OXCT1 and ACAT1. Additionally, spermidine activates the mitochondrial trifunctional protein, enhancing its thiolase activity and antitumor immunity. These regulatory mechanisms ensure that acetyl-CoA C-acyltransferase activity is tightly coupled to cellular energy demands.
acetyl-CoA C-acyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| HADHA/HADHB | LCHADD-associated chorioretinopathy | Knockout cell model (e.g., HEK293T) |
| ACAA2 | Hepatic lipid dysregulation and drug-induced liver injury | ACAA2 knockout hepatocytes |
| ACAT1 | Cancer metabolism and ketogenesis | ACAT1 knockout cancer cell lines |
| MEF2D | Muscle ketolysis and exercise capacity | Muscle-specific MEF2Dα2 knockout mice |
| PDHA1 | Myocardial ischemia/reperfusion injury | PDHA1 overexpression in cardiomyocytes |
Metabolic Myopathies and Fatty Acid Oxidation Disorders
Deficiencies in acetyl-CoA C-acyltransferase activity, particularly due to mutations in HADHA or HADHB, cause long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a severe fatty acid oxidation disorder characterized by cardiomyopathy, neuropathy, and chorioretinopathy. Patients with LCHADD often present with metabolic crises during fasting or illness. Exercise testing in metabolic myopathies can reveal impaired fatty acid oxidation, highlighting the importance of this activity for muscle energy metabolism.
Hepatic Lipid Dysregulation and Liver Injury
ACAA2, a key enzyme with acetyl-CoA C-acyltransferase activity, modulates hepatic lipid dynamics. Disruption of ACAA2 by Diosbulbin B leads to liver injury and lipid accumulation, suggesting a role in drug-induced hepatotoxicity. In chicken embryos, ACAA2 expression is associated with lipid deposition, providing a model for studying hepatic steatosis. These findings implicate this activity in non-alcoholic fatty liver disease and related metabolic disorders.
Cancer and Antitumor Immunity
The mitochondrial trifunctional protein, which harbors acetyl-CoA C-acyltransferase activity, is activated by spermidine and improves antitumor immunity in mice. This suggests that modulating this activity could enhance immune responses against tumors. Additionally, ACAT1 is studied in cancer metabolism, where it supports ketone body utilization and may influence tumor growth.
Cardiac Ischemia/Reperfusion Injury
Proteomic analysis of myocardial ischemia/reperfusion injury revealed that Xuesaitong injection attenuates injury by elevating pyruvate dehydrogenase-mediated aerobic metabolism, which is linked to acetyl-CoA production and downstream thiolase activity. This highlights the role of acetyl-CoA C-acyltransferase activity in cardiac energy metabolism and potential therapeutic targets.
From acetyl-CoA C-acyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ACAA2 impair beta-oxidation? | ACAA2 knockout cell line (e.g., HepG2) |
| Does a point mutation in HADHB affect thiolase activity? | HADHB point-mutation knock-in cells |
| Can overexpression of ACAT1 enhance ketone body utilization? | ACAT1 overexpression in muscle cells |
| Does tagging ACAA2 with GFP affect its localization? | ACAA2 knock-in with GFP tag |
| What is the role of MEF2Dα2 in muscle ketolysis? | MEF2Dα2 knockout mouse model |
| Can CRISPR library screening identify regulators of acetyl-CoA C-acyltransferase activity? | Genome-wide CRISPR knockout library in metabolic cell lines |
How to Study the acetyl-CoA C-acyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric thiolase assay | Enzyme activity by CoA release | Kinetic characterization of purified enzymes |
| Western blot | Protein expression levels | Validation of knockout or overexpression |
| Proteomics | Global protein abundance | Identifying pathway changes in disease models |
| Metabolomics | Acyl-CoA and ketone body levels | Assessing metabolic flux |
| CRISPR knockout screening | Gene essentiality and synthetic lethality | Identifying regulators of thiolase activity |
| Fluorescence microscopy | Subcellular localization | Confirming mitochondrial targeting |
| qRT-PCR | mRNA expression | Measuring transcriptional regulation |
| Seahorse assay | Mitochondrial respiration | Linking thiolase activity to oxidative phosphorylation |
Enzymatic Activity Assays
Direct measurement of acetyl-CoA C-acyltransferase activity can be performed using spectrophotometric assays that monitor the disappearance of acetoacetyl-CoA or the formation of CoA at 303 nm. These assays are essential for validating enzyme function and kinetics in cell lysates or purified protein preparations.
Proteomics and Metabolomics
Proteomic analysis can quantify the expression of enzymes with this activity, as demonstrated in myocardial ischemia/reperfusion injury studies. Metabolomics profiling of acyl-CoA species and ketone bodies provides a functional readout of pathway flux. These approaches are powerful for identifying biomarkers and understanding metabolic rewiring.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout screens can identify genes that regulate acetyl-CoA C-acyltransferase activity or are synthetic lethal with its loss. Such screens have been used to uncover metabolic vulnerabilities in cancer and immune cells. Combining screens with metabolomic profiling can reveal novel regulatory networks.
Imaging and Localization Studies
Fluorescence microscopy of tagged enzymes (e.g., GFP-ACAA2) can reveal subcellular localization and dynamics. Mitochondrial targeting sequences can be validated using mito-trackers. These methods help confirm whether the enzyme is correctly localized to mitochondria or peroxisomes.
How CRISPR Can Be Used to Study GO:0003988 acetyl-CoA C-acyltransferase activity
Knockout
CRISPR knockout of genes encoding acetyl-CoA C-acyltransferase activity (e.g., ACAA2, ACAT1) can abolish enzyme function, leading to impaired beta-oxidation and ketogenesis. These models are invaluable for studying metabolic dependencies and drug responses. For example, ACAA2 knockout hepatocytes show lipid accumulation and increased sensitivity to Diosbulbin B.
Point Mutation
Introducing point mutations in catalytic residues (e.g., the active-site cysteine) can dissect the enzymatic mechanism and separate catalytic activity from other functions. Point-mutation knock-in cell lines can model human disease variants, such as those found in HADHB deficiency. These models help validate drug targets and understand genotype-phenotype relationships.
Knock-in
Knock-in of tagged versions (e.g., GFP, FLAG) of ACAA2 or HADHA allows for real-time tracking of localization and interaction partners. Knock-in of disease-associated mutations can recapitulate human pathology in cell models. These approaches are essential for studying protein trafficking and complex assembly.
Overexpression
Overexpression of acetyl-CoA C-acyltransferase enzymes can enhance metabolic flux and alter cellular phenotypes. For instance, ACAT1 overexpression in muscle cells increases ketone body utilization. Overexpression models are useful for gain-of-function studies and for identifying downstream metabolic effects.
How EDITGENE Supports acetyl-CoA C-acyltransferase activity Research
Researchers studying acetyl-CoA C-acyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic pathways or disease. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models, enabling functional validation and mechanistic studies.
Contact EDITGENE today to design your custom CRISPR model for acetyl-CoA C-acyltransferase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| ACAA1 Knockout HEK293 Cell Line | EDJ-KQ3991 | Human | 30 | Details Get a Quote |
| HADHB Knockout HEK293 Cell Line | EDJ-KQ4838 | Human | 3032 | Details Get a Quote |
| SCP2 Knockout HEK293 Cell Line | EDJ-KQ5722 | Human | 6342 | Details Get a Quote |
| ACAA2 Knockout HEK293 Cell Line | EDJ-KQ7051 | Human | 10449 | Details Get a Quote |
| SCP2 Knockout A-549 Cell Line | EDJ-KQ29111 | Human | 6342 | Details Get a Quote |
| SCP2 Knockout HCT 116 Cell Line | EDJ-KQ29112 | Human | 6342 | Details Get a Quote |
| SCP2 Knockout HeLa Cell Line | EDJ-KQ29113 | Human | 6342 | Details Get a Quote |
| ACAA1 Knockout A-549 Cell Line | EDJ-KQ26305 | Human | 30 | Details Get a Quote |
| ACAA1 Knockout HCT 116 Cell Line | EDJ-KQ26306 | Human | 30 | Details Get a Quote |
| ACAA1 Knockout HeLa Cell Line | EDJ-KQ26307 | Human | 30 | Details Get a Quote |
| HADHB Knockout A-549 Cell Line | EDJ-KQ27617 | Human | 3032 | Details Get a Quote |
| HADHB Knockout HCT 116 Cell Line | EDJ-KQ27618 | Human | 3032 | Details Get a Quote |
| HADHB Knockout HeLa Cell Line | EDJ-KQ27619 | Human | 3032 | Details Get a Quote |
| ACAA2 Knockout HCT 116 Cell Line | EDJ-KQ30462 | Human | 10449 | Details Get a Quote |
| ACAA2 Knockout A-549 Cell Line | EDJ-KQ31836 | Human | 10449 | Details Get a Quote |
Displaying Records 1 To 15 Of 16 Records
Frequently Asked Questions About acetyl-CoA C-acyltransferase activity
What is acetyl-CoA C-acyltransferase activity?
It is an enzymatic activity (GO:0003988) that catalyzes the reversible reaction acyl-CoA + acetyl-CoA = CoA + 3-oxoacyl-CoA, essential for fatty acid beta-oxidation and ketone body metabolism.
What genes are involved in acetyl-CoA C-acyltransferase activity?
Key genes include ACAA2, ACAT1, HADHA, HADHB, and ACAA1, which encode thiolase enzymes.
What diseases are associated with acetyl-CoA C-acyltransferase deficiency?
Deficiencies cause LCHADD, metabolic myopathies, hepatic lipid dysregulation, and may affect antitumor immunity.
How is acetyl-CoA C-acyltransferase activity measured?
It is measured using spectrophotometric assays that monitor CoA release or acetoacetyl-CoA consumption.
What is the role of ACAA2 in metabolism?
ACAA2 is a mitochondrial thiolase that catalyzes the final step of beta-oxidation and modulates hepatic lipid dynamics.
Can CRISPR be used to study acetyl-CoA C-acyltransferase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are widely used to dissect gene function and disease mechanisms.
What is the difference between ACAT1 and ACAA2?
ACAT1 is involved in ketogenesis and isoleucine degradation, while ACAA2 primarily functions in fatty acid beta-oxidation.
How does spermidine affect acetyl-CoA C-acyltransferase activity?
Spermidine activates the mitochondrial trifunctional protein, enhancing its thiolase activity and antitumor immunity.
What is the mitochondrial trifunctional protein?
It is a multienzyme complex (HADHA/HADHB) that carries multiple activities including 3-ketoacyl-CoA thiolase (acetyl-CoA C-acyltransferase).
Where can I find validated CRISPR models for thiolase genes?
EDITGENE provides custom knockout, point mutation, knock-in, and overexpression cell models for thiolase genes, with full validation and bioinformatics support.
Conclusion
Acetyl-CoA C-acyltransferase activity (GO:0003988) is a cornerstone of fatty acid beta-oxidation and ketone body metabolism, with critical roles in energy homeostasis and disease. Understanding its mechanism, regulation, and genetic underpinnings is essential for developing therapies for metabolic disorders and cancer. CRISPR-based models offer powerful tools to dissect these functions, and EDITGENE provides comprehensive services to accelerate such research.
References
- 1. Al-Habsi M et al.. 2022. Spermidine activates mitochondrial trifunctional protein and improves antitumor immunity in mice.. Science 378(6618):eabj3510 PMID: 36302005
- 2. Kumar S et al.. 2025. The muscle specific MEF2Dα2 isoform promotes muscle ketolysis and running capacity in mice.. EMBO Rep 26(21):5216-5238 PMID: 40958050
- 3. Jiang Y et al.. 2025. Disruption of Acetyl-CoA Acyltransferase 2 Resulting from Exposure to Diosbulbin B.. Chem Res Toxicol 38(7):1203-1214 PMID: 40213991
- 4. Zhao X et al.. 2017. Proteomic analysis reveals Xuesaitong injection attenuates myocardial ischemia/reperfusion injury by elevating pyruvate dehydrogenase-mediated aerobic metabolism.. Mol Biosyst 13(8):1504-1511 PMID: 28632266
- 5. Peng M et al.. 2025. Acetyl-CoA acyltransferase 2 as a metabolic modulator: Unraveling its impact on hepatic lipid dynamics in chicken embryos.. Biochim Biophys Acta Mol Cell Biol Lipids 1870(7):159674 PMID: 40738480
- 6. Tarnopolsky M. 2012. Exercise testing in metabolic myopathies.. Phys Med Rehabil Clin N Am 23(1):173-86, xii PMID: 22239882
- 7. Yamashita H et al.. 2006. Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase.. Biochim Biophys Acta 1761(1):17-23 PMID: 16476568
- 8. Pomytkina NV et al.. 2025. [LCHADD-associated chorioretinopathy (case study)].. Vestn Oftalmol 141(3):48-53 PMID: 40591384
