GO:0031956 medium-chain fatty acid-CoA ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0031956 medium-chain fatty acid-CoA ligase activity catalyzes the ATP-dependent ligation of a medium-chain fatty acid (6-12 carbons) with coenzyme A to form a medium-chain fatty acyl-CoA, AMP, and diphosphate.
• This activity is part of the xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) family, which activates both endogenous medium-chain fatty acids and xenobiotic carboxylic acids such as benzoic acid and aspirin.
• The enzyme is found in liver, kidney, and colonic mucosa, and its activity is influenced by monovalent cations in a substrate-specific manner.
• Human liver mitochondria contain a specific HXM-A form of the enzyme, which has been isolated, sequenced, and expressed as a cDNA.
• Triacsin C inhibits short-, medium-, and long-chain fatty acid:CoA ligases from human liver, providing a pharmacological tool to dissect this activity.
• ACSM1, a medium-chain acyl-CoA synthetase, is downregulated by RIPK4 and promotes oxidative stress and ferroptotic death, linking this activity to cell death pathways.
Description
Medium-chain fatty acid-CoA ligase activity (GO:0031956) is a molecular function that activates medium-chain fatty acids (6-12 carbons) by conjugating them to coenzyme A in an ATP-dependent reaction. This activation is a prerequisite for fatty acid metabolism, including beta-oxidation and lipid synthesis, and also plays a critical role in the detoxification of xenobiotic carboxylic acids such as benzoic acid and aspirin. The enzyme belongs to the xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) family, which is distinct from long-chain acyl-CoA synthetases. Researchers study this activity to understand energy homeostasis, drug metabolism, and cellular stress responses. The enzyme has been characterized in various tissues, including liver, kidney, and colonic mucosa, and its activity is modulated by monovalent cations. The human liver mitochondrial HXM-A form has been cloned and expressed, enabling detailed biochemical and structural studies. Pharmacological inhibition by triacsin C further aids in dissecting its physiological roles. Given its involvement in ferroptosis and oxidative stress, this activity is emerging as a potential target in cancer and metabolic diseases.
medium-chain fatty acid-CoA ligase activity At A Glance
| GO ID | GO:0031956 |
|---|---|
| GO term | medium-chain fatty acid-CoA ligase activity |
| Ontology | molecular_function |
| Synonym | medium-chain fatty acid activation; medium-chain fatty-acid-CoA ligase activity; medium-chain-fatty-acid-CoA ligase activity |
| Major function | ATP-dependent ligation of medium-chain fatty acids (6-12 carbons) to coenzyme A, forming medium-chain fatty acyl-CoA, AMP, and diphosphate |
| Reaction | a medium-chain fatty acid + ATP + CoA = a medium-chain fatty acyl-CoA + AMP + diphosphate |
| Substrates | Medium-chain fatty acids (6-12 carbons), ATP, CoA |
| Products | Medium-chain fatty acyl-CoA, AMP, diphosphate |
| Cofactors | Mg2+ (implied by ATP-dependent ligases), monovalent cations (substrate-specific effects) |
| Inhibitors | Triacsin C |
| Tissue distribution | Liver, kidney, colonic mucosa |
What Is GO:0031956?
GO:0031956 medium-chain fatty acid-CoA ligase activity is defined as the catalysis of the reaction: a medium-chain fatty acid + ATP + CoA = a medium-chain fatty acyl-CoA + AMP + diphosphate. A medium-chain fatty acid has an aliphatic tail containing 6 to 12 carbons. This activity enables the activation of medium-chain fatty acids for subsequent metabolic pathways and xenobiotic detoxification.
Why Is medium-chain fatty acid-CoA ligase activity Important in Cell Biology?
Medium-chain fatty acid-CoA ligase activity is essential for fatty acid metabolism and xenobiotic detoxification. It activates medium-chain fatty acids for beta-oxidation and lipid biosynthesis, and also processes xenobiotic carboxylic acids such as benzoic acid and aspirin, influencing drug clearance and toxicity. Dysregulation of this activity has been linked to oxidative stress and ferroptotic cell death, with ACSM1 downregulation promoting ferroptosis. Understanding this activity is therefore relevant to metabolic disorders, cancer, and drug metabolism research.
• Enables beta-oxidation of medium-chain fatty acids for energy production.
• Plays a key role in the detoxification of benzoic acid and aspirin.
• Influences drug pharmacokinetics by activating xenobiotic carboxylic acids.
• Modulates oxidative stress and ferroptotic cell death through ACSM1.
• Exhibits tissue-specific expression, including liver, kidney, and colonic mucosa.
• Activity is sensitive to monovalent cations, affecting substrate specificity.
• Inhibited by triacsin C, a tool for studying fatty acid activation.
• Human mitochondrial HXM-A form is structurally and functionally characterized.
• Potential target for metabolic and cancer therapies.
• Relevant to understanding species differences in fatty acid metabolism.
Molecular Mechanism of medium-chain fatty acid-CoA ligase activity
Substrate Binding and Activation
In simple terms: The enzyme grabs a medium-chain fatty acid and ATP to start the reaction.
The enzyme binds a medium-chain fatty acid (6-12 carbons) and ATP, forming a fatty acyl-AMP intermediate with the release of diphosphate. This step requires magnesium ions and is analogous to other acyl-CoA synthetases. Monovalent cations can influence substrate binding in a substrate-specific manner.
CoA Ligation and Product Release
In simple terms: CoA attaches to the fatty acid, making it ready for metabolism.
The fatty acyl-AMP intermediate reacts with coenzyme A to form medium-chain fatty acyl-CoA and AMP. The acyl-CoA product is released and can enter beta-oxidation or other metabolic pathways. The reaction is reversible under certain conditions, but physiologically favors acyl-CoA formation.
Enzyme Isoforms and Tissue Distribution
In simple terms: Different forms of the enzyme exist in different tissues.
Multiple isoforms of xenobiotic/medium-chain fatty acid:CoA ligase exist, including a human liver mitochondrial HXM-A form that has been cloned and expressed. The enzyme is also present in colonic mucosa, where it activates short- and medium-chain fatty acids. These isoforms may differ in substrate specificity and regulation.
Inhibition and Regulation
In simple terms: Certain chemicals can block the enzyme, and its activity is regulated by ions.
Triacsin C inhibits short-, medium-, and long-chain fatty acid:CoA ligases from human liver, with varying potency. Monovalent cations such as Na+ and K+ affect enzyme activity in a substrate-specific manner, suggesting allosteric regulation. The enzyme's activity can also be modulated by cellular energy status and substrate availability.
Key Genes Involved in GO:0031956 medium-chain fatty acid-CoA ligase activity
The following genes and proteins are directly associated with medium-chain fatty acid-CoA ligase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ACSM1 | Medium-chain acyl-CoA synthetase; activates medium-chain fatty acids | Downregulated by RIPK4; promotes oxidative stress and ferroptosis |
| ACSM2A | Medium-chain acyl-CoA synthetase | Potential role in fatty acid metabolism and detoxification |
| ACSM2B | Medium-chain acyl-CoA synthetase | Associated with metabolic disorders |
| ACSM3 | Medium-chain acyl-CoA synthetase | May influence lipid homeostasis |
| ACSM4 | Medium-chain acyl-CoA synthetase | Expressed in specific tissues; function under study |
| ACSM5 | Medium-chain acyl-CoA synthetase | Potential role in xenobiotic metabolism |
| HXM-A | Human liver mitochondrial medium-chain fatty acid:CoA ligase | Isolated, sequenced, and expressed; model for enzyme structure |
| XM-ligase | Xenobiotic/medium-chain fatty acid:CoA ligase | Activates benzoic acid and aspirin; detoxification |
| RIPK4 | Regulator of ACSM1 expression | Downregulates ACSM1, leading to ferroptosis |
| SLC25A20 | Carnitine-acylcarnitine translocase | Transports medium-chain acyl-CoA into mitochondria |
| CPT1A | Carnitine palmitoyltransferase 1A | Long-chain fatty acid transport; not directly medium-chain |
| ACADM | Medium-chain acyl-CoA dehydrogenase | Beta-oxidation of medium-chain acyl-CoA |
| ACADS | Short-chain acyl-CoA dehydrogenase | Beta-oxidation of short-chain acyl-CoA |
| ACADL | Long-chain acyl-CoA dehydrogenase | Beta-oxidation of long-chain acyl-CoA |
| HADHA | Trifunctional enzyme subunit alpha | Mitochondrial beta-oxidation |
| HADHB | Trifunctional enzyme subunit beta | Mitochondrial beta-oxidation |
| PPARA | Peroxisome proliferator-activated receptor alpha | Regulates fatty acid oxidation genes |
How Is medium-chain fatty acid-CoA ligase activity Regulated?
Medium-chain fatty acid-CoA ligase activity is regulated at multiple levels. Monovalent cations such as Na+ and K+ modulate enzyme activity in a substrate-specific manner, suggesting allosteric regulation. The enzyme is inhibited by triacsin C, which competes with fatty acid substrates. Expression of ACSM1 is downregulated by RIPK4, linking this activity to oxidative stress and ferroptotic signaling. Additionally, the enzyme's activity can be influenced by cellular energy status and substrate availability, as it requires ATP and CoA.
medium-chain fatty acid-CoA ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ACSM1 | Ferroptosis, oxidative stress, cancer | ACSM1 knockout or overexpression in cancer cell lines |
| ACSM2A | Metabolic disorders | Knockout in hepatocytes |
| ACSM2B | Fatty acid oxidation defects | Point mutation knock-in in cell models |
| HXM-A | Mitochondrial fatty acid metabolism | Tagged knock-in for localization studies |
| XM-ligase | Xenobiotic detoxification | Overexpression in liver cell lines |
Cancer and Ferroptosis
ACSM1, a medium-chain acyl-CoA synthetase, is downregulated by RIPK4, leading to increased oxidative stress and ferroptotic cell death. This suggests that medium-chain fatty acid-CoA ligase activity may suppress ferroptosis in cancer cells, and its loss could promote cell death. Targeting this pathway could be a therapeutic strategy in cancers with RIPK4 dysregulation.
Metabolic Disorders
Medium-chain fatty acid-CoA ligase activity is essential for fatty acid metabolism, and its dysfunction may contribute to metabolic disorders such as fatty acid oxidation defects. The enzyme's role in activating medium-chain fatty acids for beta-oxidation means that impaired activity could lead to accumulation of unmetabolized fatty acids and energy deficiency.
Drug Metabolism and Detoxification
The enzyme activates xenobiotic carboxylic acids such as benzoic acid and aspirin, facilitating their conjugation and excretion. Variations in enzyme activity could affect drug clearance and toxicity, as highlighted by pharmacokinetic studies of aspirin. This makes it a potential target for drug-drug interactions and personalized medicine.
From medium-chain fatty acid-CoA ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ACSM1 increase ferroptosis? | ACSM1 knockout cell line |
| How does a point mutation affect enzyme kinetics? | Point mutation knock-in of ACSM1 |
| Where is HXM-A localized in mitochondria? | Tagged knock-in of HXM-A |
| Does overexpression of XM-ligase enhance aspirin detoxification? | Overexpression of XM-ligase in hepatocytes |
| What is the effect of triacsin C on medium-chain acyl-CoA levels? | Wild-type cells treated with triacsin C |
| Does monovalent cation concentration alter substrate specificity? | In vitro enzyme assays with purified enzyme |
How to Study the medium-chain fatty acid-CoA ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric assay | Acyl-CoA formation | Enzyme kinetics and inhibitor screening |
| Radiometric assay | Incorporation of radiolabeled fatty acid | Activity in tissue homogenates |
| RNA-seq | Gene expression levels | Tissue-specific expression profiling |
| Western blot | Protein abundance | Validation of knockout or overexpression |
| LC-MS/MS metabolomics | Acyl-CoA and fatty acid levels | Metabolic flux analysis |
| Immunofluorescence | Subcellular localization | Mitochondrial targeting of HXM-A |
| CRISPR screening | Gene essentiality and interactions | Identify regulators of ferroptosis |
Enzymatic Activity Assays
Medium-chain fatty acid-CoA ligase activity can be measured using spectrophotometric or radiometric assays that monitor the formation of medium-chain fatty acyl-CoA from fatty acid, ATP, and CoA. These assays are typically performed with purified enzyme or tissue homogenates and can be used to assess kinetic parameters and inhibitor effects.
Gene Expression Analysis
RNA-seq and qPCR can quantify the expression of ACSM family genes and other medium-chain acyl-CoA synthetases in different tissues or under various conditions. This helps identify transcriptional regulation and splice variants.
Proteomics and Western Blotting
Western blotting with specific antibodies can detect protein levels of ACSM1 and other isoforms. Proteomics approaches can identify post-translational modifications and interacting partners.
Metabolomics
LC-MS/MS-based metabolomics can measure intracellular levels of medium-chain fatty acyl-CoAs and free fatty acids, providing a readout of enzyme activity in cells.
How CRISPR Can Be Used to Study GO:0031956 medium-chain fatty acid-CoA ligase activity
Knockout
CRISPR knockout of ACSM1 or other medium-chain acyl-CoA synthetases can abolish enzyme activity, allowing researchers to study its role in fatty acid metabolism and ferroptosis. Knockout cell lines are valuable for identifying compensatory pathways and validating drug targets.
Point Mutation
Introducing point mutations in the catalytic domain of ACSM1 can dissect substrate specificity and catalytic mechanism. For example, mutations in the ATP-binding site can render the enzyme inactive, serving as negative controls.
Knock-in
Knock-in of tagged versions of HXM-A or ACSM1 enables localization and interaction studies. Fluorescent tags allow live-cell imaging of mitochondrial targeting.
Overexpression
Overexpression of medium-chain fatty acid-CoA ligase in cell lines can increase acyl-CoA levels and enhance detoxification of xenobiotics such as aspirin. This is useful for studying gain-of-function effects and drug metabolism.
How EDITGENE Supports medium-chain fatty acid-CoA ligase activity Research
Researchers studying medium-chain fatty acid-CoA ligase activity-related genes often need to determine whether a candidate gene is causally involved in fatty acid metabolism, ferroptosis, or drug detoxification. EDITGENE provides comprehensive CRISPR-based services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for medium-chain fatty acid-CoA ligase activity research.
Frequently Asked Questions About medium-chain fatty acid-CoA ligase activity
What is medium-chain fatty acid-CoA ligase activity?
It is a molecular function (GO:0031956) that catalyzes the ATP-dependent ligation of a medium-chain fatty acid (6-12 carbons) to coenzyme A, forming a medium-chain fatty acyl-CoA, AMP, and diphosphate.
What genes are involved in medium-chain fatty acid-CoA ligase activity?
Genes include ACSM1, ACSM2A, ACSM2B, ACSM3, ACSM4, ACSM5, and the human liver mitochondrial HXM-A form.
What is the reaction catalyzed by GO:0031956?
The reaction is: a medium-chain fatty acid + ATP + CoA = a medium-chain fatty acyl-CoA + AMP + diphosphate.
How is medium-chain fatty acid-CoA ligase activity regulated?
It is regulated by monovalent cations, inhibited by triacsin C, and ACSM1 expression is downregulated by RIPK4.
What diseases are associated with medium-chain fatty acid-CoA ligase activity?
It is linked to ferroptosis, oxidative stress, metabolic disorders, and drug metabolism variations.
What is the role of ACSM1 in ferroptosis?
ACSM1 downregulation by RIPK4 promotes oxidative stress and ferroptotic cell death.
How can I study medium-chain fatty acid-CoA ligase activity in the lab?
Use enzymatic assays, CRISPR knockout/knock-in models, RNA-seq, metabolomics, and Western blotting.
What is triacsin C and how does it affect this enzyme?
Triacsin C is an inhibitor of short-, medium-, and long-chain fatty acid:CoA ligases, used to study fatty acid activation.
Which tissues express medium-chain fatty acid-CoA ligase?
It is expressed in liver, kidney, and colonic mucosa.
What is the HXM-A form?
HXM-A is a human liver mitochondrial medium-chain fatty acid:CoA ligase that has been isolated, sequenced, and expressed.
Conclusion
Medium-chain fatty acid-CoA ligase activity (GO:0031956) is a critical molecular function for fatty acid metabolism and xenobiotic detoxification. Its role in ferroptosis and drug metabolism makes it a promising target for cancer and metabolic disease research. Understanding its regulation and developing precise CRISPR models will advance therapeutic strategies. EDITGENE provides the tools to study this activity in relevant cellular contexts.
References
- 1. Zhang J et al.. 2024. RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1.. Proc Natl Acad Sci U S A 121(40):e2410628121 PMID: 39316049
- 2. van der Sluis R et al.. 2016. Xenobiotic/medium chain fatty acid: CoA ligase - a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin.. Expert Opin Drug Metab Toxicol 12(10):1169-79 PMID: 27351777
- 3. Vessey DA et al.. 2000. Monovalent cation effects on the activity of the xenobiotic/medium-chain fatty acid:CoA ligases are substrate specific.. J Biochem Mol Toxicol 14(3):162-8 PMID: 10711632
- 4. Vessey DA. 2001. Isolation and preliminary characterization of the medium-chain fatty acid:CoA ligase responsible for activation of short- and medium-chain fatty acids in colonic mucosa from swine.. Dig Dis Sci 46(2):438-42 PMID: 11281196
- 5. Vessey DA et al.. 2003. Isolation, sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria.. J Biochem Mol Toxicol 17(1):1-6 PMID: 12616642
- 6. Tol VA. 1975. Aspects of long-chain acyl-COA metabolism.. Mol Cell Biochem 7(1):19-31 PMID: 1134497
- 7. Visagie JL et al.. 2024. Pharmacokinetics of aspirin: evaluating shortcomings in the literature.. Expert Opin Drug Metab Toxicol 20(8):727-740 PMID: 39092921
- 8. Vessey DA et al.. 2004. Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver.. J Biochem Mol Toxicol 18(2):100-6 PMID: 15122652
