GO:0016877 ligase activity, forming carbon-sulfur bonds: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016877 describes the catalytic joining of two molecules via a carbon-sulfur bond, coupled to ATP or similar triphosphate hydrolysis.
• This activity is essential for xenobiotic and aromatic compound degradation, coenzyme A (CoA) metabolism, and sulfur assimilation.
• Isophthalate:coenzyme A ligase is a well-characterized enzyme that initiates anaerobic degradation of the xenobiotic isophthalate.
• The reaction proceeds through adenylation of a carboxylate substrate, followed by nucleophilic attack by a thiol (e.g., CoA) to form a thioester.
• Dysregulation of carbon-sulfur bond-forming ligases is linked to metabolic disorders and microbial pathogenesis.
• CRISPR knockout, point mutation, knock-in, and overexpression models enable precise functional dissection of these enzymes.
Description
GO:0016877, ligase activity, forming carbon-sulfur bonds, is a molecular function ontology term that defines enzymes catalyzing the ATP-dependent joining of two molecules through a carbon-sulfur bond. These ligases are fundamental to cellular metabolism, enabling the activation of carboxylate substrates for subsequent thioesterification with coenzyme A (CoA) or other thiols. This activity is critical for the degradation of aromatic xenobiotics, fatty acid metabolism, and the biosynthesis of sulfur-containing biomolecules. Researchers study these enzymes to understand microbial catabolic pathways, metabolic engineering, and the molecular basis of diseases linked to sulfur metabolism. The isophthalate:coenzyme A ligase exemplifies how a carbon-sulfur bond-forming ligase initiates the anaerobic degradation of a xenobiotic compound, highlighting the environmental and biotechnological relevance of this enzyme class.
ligase activity, forming carbon-sulfur bonds At A Glance
| GO ID | GO:0016877 |
|---|---|
| GO term | ligase activity, forming carbon-sulfur bonds |
| Ontology | molecular_function |
| Synonym | ligase activity, forming carbon-sulphur bonds |
| Major function | Catalysis of carbon-sulfur bond formation with concomitant ATP hydrolysis |
| EC number | 6.2.1.- |
| Reaction | ATP + substrate + thiol → AMP + diphosphate + thioester |
| Substrates | Carboxylate-containing compounds (e.g., isophthalate) and thiols (e.g., CoA) |
| Cofactors | Mg2+ or Mn2+ (typical for adenylating enzymes) |
| Localization | Cytoplasm, mitochondria, peroxisomes (varies by enzyme) |
What Is GO:0016877?
In our own words, GO:0016877 encompasses enzymes that catalyze the formation of a carbon-sulfur bond between two substrates, using the energy released by hydrolysis of the diphosphate bond in ATP or a similar triphosphate. This definition captures a broad class of ligases that activate a carboxylate group via adenylation, then transfer the acyl group to a thiol, producing a thioester and releasing AMP and pyrophosphate.
Why Is ligase activity, forming carbon-sulfur bonds Important in Cell Biology?
GO:0016877 is important because carbon-sulfur bond-forming ligases are central to the activation and detoxification of xenobiotic compounds, the metabolism of fatty acids and aromatic acids, and the biosynthesis of cofactors and secondary metabolites. Their activity determines the fate of environmental pollutants and drugs, and their dysfunction can lead to metabolic imbalances and disease.
• Enables anaerobic degradation of xenobiotic isophthalate, a model for aromatic pollutant breakdown.
• Essential for coenzyme A (CoA) metabolism and fatty acid activation.
• Contributes to sulfur assimilation and biosynthesis of sulfur-containing amino acids and vitamins.
• Plays a role in microbial pathogenesis and host-microbe interactions.
• Target for metabolic engineering to produce value-added chemicals from aromatic compounds.
• Involved in drug metabolism and detoxification pathways.
• Dysregulation linked to metabolic disorders and cancer.
• Provides a mechanistic basis for designing enzyme inhibitors and prodrugs.
• Facilitates the study of enzyme evolution and catalytic promiscuity.
• Supports biotechnological applications in bioremediation and green chemistry.
What Happens During ligase activity, forming carbon-sulfur bonds?
Substrate Binding and Adenylation
In simple terms: The enzyme grabs the substrate and attaches AMP to it using ATP.
The catalytic cycle begins with the binding of a carboxylate substrate (e.g., isophthalate) and ATP to the enzyme active site. In the presence of Mg2+, the enzyme catalyzes the adenylation of the substrate, forming an acyl-adenylate intermediate and releasing pyrophosphate. This step activates the carboxylate for nucleophilic attack.
Thioester Formation
In simple terms: A sulfur-containing molecule like CoA attacks the activated substrate, forming a carbon-sulfur bond.
The activated acyl-adenylate is then attacked by a thiol (e.g., coenzyme A) at the carbonyl carbon, displacing AMP and forming a thioester bond between the substrate and the thiol. This carbon-sulfur bond formation is the defining feature of GO:0016877.
Product Release and Enzyme Turnover
In simple terms: The thioester product leaves the enzyme, and the enzyme is ready for another round.
After thioester formation, the product (e.g., isophthalyl-CoA) is released from the active site, and the enzyme undergoes conformational changes to reset for the next catalytic cycle. The overall reaction consumes ATP and produces AMP and pyrophosphate.
Physiological Context
In simple terms: This reaction happens in cells to break down or build molecules that contain sulfur bonds.
In vivo, this activity is integrated into pathways such as anaerobic aromatic degradation, where isophthalate:coenzyme A ligase initiates the catabolism of isophthalate by converting it to isophthalyl-CoA, which then enters further degradation steps. This allows microorganisms to utilize xenobiotic compounds as carbon sources.
Key Genes Involved in GO:0016877 ligase activity, forming carbon-sulfur bonds
The following genes encode enzymes with ligase activity forming carbon-sulfur bonds, as exemplified by isophthalate:coenzyme A ligase and related CoA ligases.
| Gene | Major Role | Research Relevance |
|---|---|---|
| iphl | Isophthalate:CoA ligase; initiates anaerobic isophthalate degradation | Model for xenobiotic degradation and aromatic acid metabolism |
| fadD | Long-chain fatty acyl-CoA ligase | Fatty acid activation and beta-oxidation |
| acs | Acetyl-CoA synthetase | Central carbon metabolism and acetate utilization |
| 4CL | 4-coumarate:CoA ligase | Phenylpropanoid biosynthesis in plants |
| luciferase | Firefly luciferase (acyl-CoA ligase family) | Bioluminescence and reporter assays |
| menE | o-succinylbenzoate:CoA ligase | Menaquinone biosynthesis |
| dhbE | 2,3-dihydroxybenzoate-AMP ligase | Siderophore biosynthesis |
| entE | 2,3-dihydroxybenzoate-AMP ligase | Enterobactin synthesis |
| basE | 2,3-dihydroxybenzoate-AMP ligase | Bacillibactin synthesis |
| paaK | Phenylacetate:CoA ligase | Phenylacetate degradation |
| hcaD | Hydroxycinnamate:CoA ligase | Aromatic compound degradation |
| bcrA | Benzoate:CoA ligase | Anaerobic benzoate degradation |
| bamB | Benzylsuccinate:CoA ligase | Toluene degradation |
| sucC | Succinyl-CoA synthetase beta subunit | TCA cycle and carbon-sulfur bond formation |
| sucD | Succinyl-CoA synthetase alpha subunit | TCA cycle and carbon-sulfur bond formation |
| lsc1 | Succinyl-CoA ligase subunit | Mitochondrial metabolism |
| lsc2 | Succinyl-CoA ligase subunit | Mitochondrial metabolism |
How Is ligase activity, forming carbon-sulfur bonds Regulated?
The activity of carbon-sulfur bond-forming ligases is regulated at multiple levels. In bacteria, expression of genes like iphl is induced by the presence of substrates such as isophthalate, often under anaerobic conditions. Post-translational modifications, such as phosphorylation, can modulate enzyme activity. Additionally, feedback inhibition by downstream metabolites (e.g., CoA derivatives) may control flux through these pathways.
ligase activity, forming carbon-sulfur bonds and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SUCLA2 | Mitochondrial DNA depletion syndrome | Knockout in HEK293 cells; patient-derived fibroblasts |
| SUCLG1 | Encephalomyopathy | Knock-in of patient mutations in iPSCs |
| ACSL4 | Cancer, ferroptosis | Overexpression in cancer cell lines; CRISPR KO |
| entE | Bacterial virulence | Knockout in E. coli; infection models |
| iphl | Xenobiotic degradation | Knockout in anaerobic bacteria; bioremediation studies |
Metabolic Disorders
Defects in carbon-sulfur bond-forming ligases can lead to metabolic imbalances. For example, mutations in succinyl-CoA ligase subunits (SUCLA2, SUCLG1) cause mitochondrial DNA depletion syndromes and encephalomyopathies. These enzymes are essential for the TCA cycle and provide succinyl-CoA for heme synthesis.
Cancer
Altered expression of acyl-CoA ligases has been observed in various cancers, where they contribute to lipid metabolism reprogramming and tumor growth. Targeting these enzymes is being explored as a therapeutic strategy.
Infectious Diseases
Bacterial ligases involved in siderophore biosynthesis (e.g., entE, dhbE) are virulence factors. Inhibiting these enzymes could disarm pathogens by blocking iron acquisition.
Neurological Disorders
Impaired mitochondrial fatty acid activation due to defects in acyl-CoA ligases can cause neuropathies and neurodegeneration. The brain relies heavily on fatty acid metabolism for energy.
From ligase activity, forming carbon-sulfur bonds-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does gene X have carbon-sulfur bond-forming ligase activity? | In vitro enzymatic assay with purified protein |
| What is the role of gene X in isophthalate degradation? | CRISPR knockout in Desulfobacterium sp. |
| How does a point mutation affect catalytic efficiency? | Site-directed mutagenesis and kinetic analysis |
| Can gene X be used as a reporter? | Knock-in of luciferase into a target locus |
| Does overexpression of gene X alter lipid metabolism? | Overexpression in HepG2 cells followed by lipidomics |
| What is the interactome of gene X? | Tagged knock-in (e.g., FLAG) and immunoprecipitation |
How to Study the ligase activity, forming carbon-sulfur bonds Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme kinetics | Catalytic efficiency (kcat/Km) | Characterizing wild-type vs mutant ligases |
| CRISPR knockout | Loss-of-function phenotype | Pathway elucidation |
| RNA-seq | Gene expression changes | Identifying co-regulated genes |
| Proteomics | Protein abundance and modifications | Validating knockout effects |
| Immunoprecipitation | Protein interactions | Discovering binding partners |
| Metabolomics | Metabolite levels | Assessing pathway flux |
| Reporter assays | Promoter activity | Studying gene regulation |
Enzymatic Assays
Direct measurement of ligase activity using spectrophotometric or chromatographic methods. For example, isophthalate:CoA ligase activity can be monitored by the formation of isophthalyl-CoA using HPLC or by coupling to a CoA-consuming reaction.
CRISPR-Cas9 Knockout
Generating knockout cell lines or bacterial strains to study loss-of-function phenotypes. This is particularly useful for assessing the role of ligases in metabolic pathways.
RNA Sequencing (RNA-seq)
Transcriptomic profiling to identify genes co-regulated with carbon-sulfur bond-forming ligases under various conditions, revealing pathway context.
Proteomics and Interactomics
Mass spectrometry-based approaches to identify protein-protein interactions and post-translational modifications of ligases, using tagged knock-in models.
How CRISPR Can Be Used to Study GO:0016877 ligase activity, forming carbon-sulfur bonds
Knockout
CRISPR knockout of genes encoding carbon-sulfur bond-forming ligases (e.g., iphl) can abolish the ability to degrade specific substrates, providing direct evidence of their function. Knockout cell lines are also used to study metabolic reprogramming in cancer.
Point Mutation
Introducing point mutations in catalytic residues (e.g., the adenylation domain) via CRISPR base editing or homology-directed repair allows precise dissection of the enzymatic mechanism and its physiological consequences.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins enables visualization and purification of ligases for interaction studies and localization. Knock-in of disease-associated mutations creates isogenic models.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of ligases can reveal gain-of-function phenotypes, such as enhanced xenobiotic degradation or altered lipid metabolism.
How EDITGENE Supports ligase activity, forming carbon-sulfur bonds Research
Researchers studying ligase activity, forming carbon-sulfur bonds-related genes often need to determine whether a candidate gene is causally involved in a specific metabolic or disease pathway. EDITGENE provides a comprehensive suite of CRISPR services to accelerate this discovery.
Contact EDITGENE today to design your custom CRISPR model for ligase activity, forming carbon-sulfur bonds research.
Frequently Asked Questions About ligase activity, forming carbon-sulfur bonds
What is GO:0016877?
GO:0016877 is a Gene Ontology molecular function term for ligase activity that forms carbon-sulfur bonds, coupled to ATP hydrolysis.
What genes are involved in ligase activity, forming carbon-sulfur bonds?
Genes include iphl, fadD, acs, 4CL, menE, dhbE, entE, and succinyl-CoA ligase subunits.
What is the function of isophthalate:coenzyme A ligase?
It initiates anaerobic isophthalate degradation by forming isophthalyl-CoA, a carbon-sulfur bond-containing intermediate.
How is carbon-sulfur bond formation catalyzed?
Via adenylation of a carboxylate substrate followed by thiol attack, forming a thioester.
What diseases are associated with defects in carbon-sulfur bond-forming ligases?
Mutations in succinyl-CoA ligase cause mitochondrial DNA depletion syndromes; other ligases are linked to cancer and metabolic disorders.
What experimental models are used to study GO:0016877?
CRISPR knockout, point mutation, knock-in, and overexpression models in cell lines and bacteria.
How can I measure ligase activity forming carbon-sulfur bonds?
Using enzymatic assays that detect thioester formation, often coupled to CoA consumption.
What is the role of ATP in GO:0016877?
ATP provides energy by hydrolysis of its diphosphate bond to drive carbon-sulfur bond formation.
Are there inhibitors of carbon-sulfur bond-forming ligases?
Some inhibitors target adenylation domains; they are studied for antibacterial and anticancer therapy.
How does EDITGENE support research on GO:0016877?
EDITGENE offers CRISPR knockout, point mutation, knock-in, overexpression, library screening, and bioinformatics services.
Conclusion
GO:0016877, ligase activity, forming carbon-sulfur bonds, represents a vital enzymatic function in metabolism, xenobiotic degradation, and disease. Understanding its mechanism and regulation opens avenues for therapeutic intervention and biotechnological applications. EDITGENE's CRISPR services empower researchers to dissect these pathways with precision.
References
- 1. Junghare M et al.. 2022. Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate.. BMC Microbiol 22(1):227 PMID: 36171563
