GO:0016879 ligase activity, forming carbon-nitrogen bonds: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016879 describes a molecular function: the ATP-dependent joining of two molecules or two groups within a single molecule via a carbon-nitrogen bond.
• This activity is essential in bacterial metabolism, and enzymes annotated with this function are enriched among essential genes in bacteria.
• A well-studied example is 5,10-methenyltetrahydrofolate synthetase (MTHFS), which catalyzes the ATP-dependent cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, forming a carbon-nitrogen bond.
• The ATP binding site of MTHFS is critical for catalysis, and mutations in specific amino acids can abolish or alter enzyme activity.
• Biotin carboxylase, another enzyme with this activity, is a target for inhibitor design, and computational redesign of inhibitors has been explored.
• Studying GO:0016879 helps researchers understand essential metabolic pathways and develop antimicrobial or anticancer strategies [1,3].
Description
GO:0016879, ligase activity, forming carbon-nitrogen bonds, is a molecular function that catalyzes the ATP-dependent formation of carbon-nitrogen bonds. This activity is fundamental to many biosynthetic pathways, including the synthesis of amino acids, nucleotides, and cofactors. Enzymes with this activity are widespread across all domains of life and are often essential for cellular survival. In bacteria, genes encoding such enzymes are significantly enriched among essential genes, highlighting their potential as antibacterial targets. For example, 5,10-methenyltetrahydrofolate synthetase (MTHFS) catalyzes the formation of a carbon-nitrogen bond in folate metabolism, a pathway critical for one-carbon transfer reactions. Another example is biotin carboxylase, which catalyzes the ATP-dependent carboxylation of biotin, a key step in fatty acid biosynthesis and a target for inhibitor design. Understanding the mechanism and regulation of these enzymes is vital for basic biology and drug discovery.
ligase activity, forming carbon-nitrogen bonds At A Glance
| GO ID | GO:0016879 |
|---|---|
| GO term | ligase activity, forming carbon-nitrogen bonds |
| Ontology | molecular_function |
| Synonym | other carbon-nitrogen ligase activity |
| Major function | ATP-dependent formation of carbon-nitrogen bonds |
| EC number | 6.3.-.- |
| Examples | MTHFS, biotin carboxylase, and other ligases |
| Cofactors | ATP or similar triphosphate |
| Reaction | Joining of two molecules via C-N bond with ATP hydrolysis |
What Is GO:0016879?
According to the Gene Ontology, GO:0016879 is defined as the catalysis of the joining of two molecules, or two groups within a single molecule, via a carbon-nitrogen bond, with the concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate. In simpler terms, it is an enzyme activity that uses ATP to link molecules together by forming a carbon-nitrogen bond. This definition encompasses a variety of ligases that activate a substrate through adenylation or phosphorylation, followed by nucleophilic attack by an amine group, resulting in bond formation.
Why Is ligase activity, forming carbon-nitrogen bonds Important in Cell Biology?
GO:0016879 is important because it underpins essential metabolic processes such as folate metabolism, fatty acid biosynthesis, and amino acid synthesis [1,2,3]. Disruption of these enzymes can lead to growth arrest or cell death, making them attractive targets for antibiotics and cancer therapeutics [1,3]. Moreover, understanding the catalytic mechanism and regulation of these enzymes can inform the design of specific inhibitors and guide metabolic engineering.
• Enzymes with this activity are enriched in bacterial essential genes, indicating their potential as broad-spectrum antibacterial targets.
• MTHFS, a representative enzyme, is crucial for folate metabolism and one-carbon transfer, impacting nucleotide synthesis and methylation.
• Biotin carboxylase is a key enzyme in fatty acid biosynthesis and a validated target for herbicides and antibiotics.
• Defects in carbon-nitrogen ligases can lead to metabolic disorders and are implicated in cancer and neurodegeneration [2,3].
• These enzymes are often regulated by feedback inhibition and post-translational modifications, affecting pathway flux.
• Studying their mechanism aids in the development of mechanism-based inhibitors.
• They are potential targets for anti-infective and anticancer drug discovery [1,3].
• Understanding their structure-function relationships enables protein engineering and synthetic biology applications [2,3].
Molecular Mechanism of ligase activity, forming carbon-nitrogen bonds
Substrate Binding and Activation
In simple terms: The enzyme grabs the substrate and uses ATP to make it more reactive.
The catalytic cycle begins with the binding of substrates to the enzyme active site. For example, in 5,10-methenyltetrahydrofolate synthetase (MTHFS), the substrate 5-formyltetrahydrofolate binds, and ATP is positioned for phosphoryl transfer. The ATP binding site contains specific amino acids that are critical for catalysis, as shown by mutational studies. Similarly, biotin carboxylase binds biotin and ATP, with the ATP binding site being a target for inhibitor design.
Formation of Activated Intermediate
In simple terms: ATP is used to add a phosphate group to the substrate, making it ready to react.
Upon substrate binding, the enzyme catalyzes the transfer of the gamma-phosphate from ATP to the substrate, forming an activated intermediate such as an acyl phosphate or phosphorylated substrate. In MTHFS, this step leads to the formation of 5,10-methenyltetrahydrofolate through cyclization, forming a carbon-nitrogen bond. The hydrolysis of ATP to ADP and inorganic phosphate provides the energy for this activation.
Carbon-Nitrogen Bond Formation
In simple terms: The activated substrate reacts with an amine group to form a new carbon-nitrogen bond.
The activated intermediate undergoes nucleophilic attack by an amine group, resulting in the formation of a carbon-nitrogen bond. In MTHFS, the amine group of the substrate attacks the carbonyl carbon, leading to ring closure and formation of the product. This step is often rate-limiting and is tightly regulated. In biotin carboxylase, the carboxylation of biotin involves the formation of a carbon-nitrogen bond between the ureido nitrogen and carbon dioxide, although the exact mechanism may vary.
Product Release and Enzyme Turnover
In simple terms: The product is released, and the enzyme is ready for another round.
After bond formation, the product is released from the active site, and the enzyme returns to its initial state. Conformational changes may facilitate product release and reset the active site for the next catalytic cycle. For MTHFS, the product 5,10-methenyltetrahydrofolate is released, and the enzyme can bind new substrate. The overall reaction is energetically driven by ATP hydrolysis.
Cofactors and Regulation
In simple terms: Some enzymes need helper molecules and are controlled by cellular signals.
Many enzymes with this activity require divalent metal ions such as Mg2+ for ATP binding and catalysis. For instance, MTHFS activity is dependent on Mg2+. Regulation can occur through feedback inhibition by downstream metabolites, as seen in folate metabolism where 5,10-methenyltetrahydrofolate may inhibit upstream enzymes. Additionally, post-translational modifications and allosteric regulation can modulate activity [2,3].
Key Genes Involved in GO:0016879 ligase activity, forming carbon-nitrogen bonds
The following genes encode enzymes that exhibit ligase activity forming carbon-nitrogen bonds, as supported by experimental evidence and annotations.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTHFS | Catalyzes conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate | Folate metabolism, one-carbon transfer, potential anticancer target |
| ACC | Biotin carboxylase component of acetyl-CoA carboxylase | Fatty acid biosynthesis, target for inhibitors |
| GlnA | Glutamine synthetase, forms glutamine from glutamate and ammonia | Nitrogen metabolism, essential in bacteria |
| PurD | Phosphoribosylamine-glycine ligase, purine biosynthesis | Nucleotide synthesis, antibacterial target |
| PurT | Phosphoribosylglycinamide formyltransferase 2 | Purine biosynthesis, essential in E. coli |
| CysE | Serine acetyltransferase, forms cysteine from serine | Cysteine biosynthesis, antibacterial target |
| AsnB | Asparagine synthetase B, forms asparagine from aspartate | Amino acid metabolism, essential in some bacteria |
| GatA | Glutamyl-tRNA(Gln) amidotransferase subunit A | Protein synthesis, essential in bacteria |
| GatB | Glutamyl-tRNA(Gln) amidotransferase subunit B | Protein synthesis, essential in bacteria |
| GatC | Glutamyl-tRNA(Gln) amidotransferase subunit C | Protein synthesis, essential in bacteria |
| MurC | UDP-N-acetylmuramate-alanine ligase | Peptidoglycan biosynthesis, antibacterial target |
| MurD | UDP-N-acetylmuramoylalanine-D-glutamate ligase | Peptidoglycan biosynthesis, antibacterial target |
| MurE | UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase | Peptidoglycan biosynthesis, antibacterial target |
| MurF | UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase | Peptidoglycan biosynthesis, antibacterial target |
| Ddl | D-alanine-D-alanine ligase | Peptidoglycan biosynthesis, antibacterial target |
| FolC | Dihydrofolate synthase/folylpolyglutamate synthase | Folate metabolism, antibacterial target |
| CobB | Cobyrinic acid a,c-diamide synthase | Cobalamin biosynthesis, essential in some bacteria |
How Is ligase activity, forming carbon-nitrogen bonds Regulated?
The activity of enzymes forming carbon-nitrogen bonds is regulated at multiple levels. For MTHFS, feedback inhibition by 5,10-methenyltetrahydrofolate and regulation by the availability of substrates and cofactors such as Mg2+ have been reported. In biotin carboxylase, allosteric regulation by acetyl-CoA and phosphorylation may modulate activity. Additionally, gene expression of these enzymes can be controlled by transcription factors responsive to metabolic needs, as seen in bacterial essential genes.
ligase activity, forming carbon-nitrogen bonds and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTHFS | Folate metabolism disorders, cancer | Knockout cell lines, point mutations in ATP binding site |
| ACC | Obesity, diabetes, cancer | Knockout mice, overexpression in adipocytes |
| MurC | Bacterial infections | Bacterial knockout, inhibitor screening |
| Ddl | Bacterial infections | Bacterial knockout, inhibitor screening |
| FolC | Bacterial infections, folate metabolism | Bacterial knockout, inhibitor screening |
Cancer and Folate Metabolism
MTHFS is involved in folate metabolism, which is critical for nucleotide synthesis and methylation. Dysregulation of folate pathways can contribute to cancer development, and MTHFS has been explored as a potential target for anticancer therapy.
Bacterial Infections and Antibiotic Resistance
Many enzymes with carbon-nitrogen ligase activity are essential in bacteria, including those involved in peptidoglycan and folate biosynthesis. Inhibitors of these enzymes, such as biotin carboxylase inhibitors, are being developed as novel antibiotics [1,3].
Metabolic Disorders
Deficiencies in enzymes like MTHFS can lead to metabolic imbalances, including neurological symptoms due to impaired folate metabolism.
From ligase activity, forming carbon-nitrogen bonds-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of MTHFS knockout on cell proliferation? | CRISPR knockout in cancer cell lines |
| How do point mutations in ATP binding site affect MTHFS activity? | Point mutation knock-in in cell lines |
| Can biotin carboxylase inhibitors be redesigned for better efficacy? | Structure-based virtual screening and knock-in models |
| Is MurC essential in bacteria? | CRISPR interference or knockout in bacteria |
| What is the role of Ddl in antibiotic resistance? | Overexpression and knockout in bacterial strains |
| How does FolC contribute to folate metabolism? | Knockout and overexpression in bacterial cells |
How to Study the ligase activity, forming carbon-nitrogen bonds Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | Catalytic activity | Measure MTHFS or biotin carboxylase activity [2,3] |
| Site-directed mutagenesis | Effect of specific mutations on activity | Identify critical residues in ATP binding site |
| X-ray crystallography | Three-dimensional structure | Determine enzyme-inhibitor complexes |
| Virtual screening | Binding affinity of compounds | Discover new inhibitors |
| CRISPR knockout | Gene function | Assess essentiality in bacteria or cancer cells |
| RNA-seq | Gene expression changes | Study regulation of ligase genes |
| Proteomics | Protein abundance and modifications | Analyze post-translational regulation |
| Metabolomics | Metabolite levels | Measure pathway flux |
Enzymatic Assays
Enzymatic activity of carbon-nitrogen ligases can be measured using spectrophotometric or radiometric assays that monitor ATP hydrolysis or product formation. For MTHFS, activity is often assayed by following the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate at 350 nm.
Mutagenesis and Kinetic Studies
Site-directed mutagenesis of key residues in the ATP binding site, followed by kinetic analysis, can reveal essential amino acids for catalysis. For example, mutations in MTHFS ATP binding site altered Km and kcat values.
Structural Biology
X-ray crystallography and cryo-EM can provide insights into the three-dimensional structure of these enzymes and their complexes with substrates and inhibitors. Structures of biotin carboxylase have guided inhibitor design.
Computational Docking and Virtual Screening
Structure-based virtual screening of combinatorial libraries can identify novel inhibitors of enzymes like biotin carboxylase. This approach has been used to redesign inhibitors with improved properties.
How CRISPR Can Be Used to Study GO:0016879 ligase activity, forming carbon-nitrogen bonds
Knockout
CRISPR knockout can be used to delete genes encoding carbon-nitrogen ligases to study their essentiality. For example, knockout of MTHFS in cancer cell lines can reveal its role in folate metabolism and cell survival. In bacteria, knockout of essential ligase genes like MurC results in growth arrest, validating them as drug targets.
Point Mutation
Point mutations can be introduced into the ATP binding site of MTHFS to dissect the catalytic mechanism. For instance, mutating specific amino acids identified in the ATP binding site can abolish activity, as shown by biochemical studies. CRISPR-based base editing can create such point mutations in endogenous loci.
Knock-in
Knock-in of tagged versions of ligase genes (e.g., GFP or FLAG) allows for localization and interaction studies. For example, knocking in a tagged MTHFS can help track its expression and subcellular localization. Knock-in of disease-associated mutations can model metabolic disorders.
Overexpression
Overexpression of carbon-nitrogen ligases can be achieved by CRISPR activation or by introducing a constitutive promoter. Overexpression of biotin carboxylase in bacterial cells can increase fatty acid production, useful for metabolic engineering. Overexpression of MTHFS can lead to altered folate metabolism and may promote cell proliferation.
How EDITGENE Supports ligase activity, forming carbon-nitrogen bonds Research
Researchers studying ligase activity, forming carbon-nitrogen bonds-related genes often need to determine whether a candidate gene is causally involved in a specific pathway or disease. EDITGENE provides a comprehensive suite of CRISPR services to enable precise genetic modifications, from knockout to knock-in, facilitating functional studies and drug target validation.
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Frequently Asked Questions About ligase activity, forming carbon-nitrogen bonds
What is GO:0016879?
GO:0016879 is a Gene Ontology molecular function term that describes ligase activity forming carbon-nitrogen bonds, catalyzing the ATP-dependent joining of two molecules via a carbon-nitrogen bond.
What genes are involved in ligase activity, forming carbon-nitrogen bonds?
Genes include MTHFS, ACC, GlnA, PurD, MurC, Ddl, and FolC, among others, as identified in bacterial essential gene studies [1,2,3].
What is the function of MTHFS?
MTHFS (5,10-methenyltetrahydrofolate synthetase) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, forming a carbon-nitrogen bond.
How is ligase activity, forming carbon-nitrogen bonds regulated?
Regulation occurs through feedback inhibition, substrate availability, and post-translational modifications, as seen in MTHFS and biotin carboxylase [2,3].
What diseases are associated with defects in carbon-nitrogen ligases?
Defects can lead to metabolic disorders, cancer, and increased susceptibility to bacterial infections [1,2,3].
What methods are used to study ligase activity, forming carbon-nitrogen bonds?
Methods include enzymatic assays, mutagenesis, structural biology, and computational screening [2,3].
How can CRISPR be used to study these enzymes?
CRISPR knockout, point mutation, knock-in, and overexpression enable functional studies and drug target validation [1,2,3].
Why are bacterial carbon-nitrogen ligases important for antibiotic development?
Many are essential for bacterial survival and are enriched among essential genes, making them attractive antibiotic targets.
What is the role of ATP in ligase activity, forming carbon-nitrogen bonds?
ATP provides energy through hydrolysis of its diphosphate bond to drive the formation of the carbon-nitrogen bond.
Can inhibitors of carbon-nitrogen ligases be designed?
Yes, structure-based virtual screening has been used to redesign inhibitors of biotin carboxylase, a representative enzyme.
Conclusion
GO:0016879, ligase activity forming carbon-nitrogen bonds, represents a fundamental enzymatic function essential for metabolism and cell survival. Its role in bacterial essential genes and human metabolic pathways makes it a promising target for antibiotics and cancer therapy [1,2,3]. Continued research into the mechanism, regulation, and inhibition of these enzymes will provide insights into basic biology and facilitate drug discovery. EDITGENE's CRISPR services empower researchers to precisely manipulate these genes and accelerate their studies.
References
- 1. Gao F et al.. 2011. Enzymes are enriched in bacterial essential genes.. PLoS One 6(6):e21683 PMID: 21738765
- 2. Tolley M et al.. 2012. Investigations of amino acids in the ATP binding site of 5,10-methenyltetrahydrofolate synthetase.. Protein J 31(6):519-28 PMID: 22773193
- 3. Brylinski M et al.. 2014. Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.. Molecules 19(4):4021-45 PMID: 24699146
