GO:0106408 diadenylate cyclase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106408 diadenylate cyclase activity catalyzes the reaction 2 ATP = 3',3'-c-di-AMP + 2 diphosphate, producing the second messenger cyclic di-AMP.
• Diadenylate cyclases (DACs) are defined by a DisA_N (DAC) domain and occur in diverse bacterial and archaeal domain architectures.
• The founding enzyme DisA from Thermotoga maritima is a DNA recombination intermediate-binding checkpoint protein whose cyclase activity is regulated by DNA.
• DAC activity is controlled by autoinhibitory domains, as shown for CdaS in Bacillus subtilis, and by the availability of the ATP substrate pool.
• Small molecules such as polyphenols and IPA-3 can inhibit diadenylate cyclases, providing chemical tools and antimicrobial leads.
• In Streptococcus suis, the diadenylate cyclase ssDacA contributes to c-di-AMP production and is a validated target for inhibitor development.
Description
Diadenylate cyclase activity (GO:0106408) is the enzymatic activity that synthesizes the bacterial second messenger cyclic di-AMP from two molecules of ATP, releasing two molecules of diphosphate. This activity is encoded by a conserved DisA_N (DAC) domain and is found across bacterial and archaeal genomes in a remarkable variety of domain architectures. Because c-di-AMP controls cell wall homeostasis, potassium transport, DNA integrity surveillance and virulence, the enzymes that produce it are central to bacterial physiology and pathogenesis. Researchers study diadenylate cyclase activity to understand how bacteria sense and respond to stress, how second messenger pools are maintained, and how these enzymes can be targeted by new antimicrobials. The activity was first structurally and biochemically characterized for the checkpoint protein DisA, which revealed that DNA recombination intermediates regulate c-di-AMP synthesis. Subsequent work identified additional DACs such as CdaS in Bacillus subtilis and ssDacA in Streptococcus suis, showing that autoinhibitory domains and substrate availability tune c-di-AMP output. More recently, a miniature CRISPR-Cas10 enzyme was shown to confer immunity through inhibitory signalling involving a diadenylate cyclase-related mechanism, linking this activity to defense systems.
diadenylate cyclase activity At A Glance
| GO ID | GO:0106408 |
|---|---|
| GO term | diadenylate cyclase activity |
| Ontology | molecular_function |
| Synonym | cyclic-di-AMP synthase |
| Definition | Catalysis of the reaction: 2 ATP = 3',3'-c-di-AMP + 2 diphosphate. |
| Reaction | 2 ATP = 3',3'-c-di-AMP + 2 diphosphate |
| Product | 3',3'-cyclic di-AMP (c-di-AMP) |
| Substrate | ATP |
| Representative domain | DisA_N (DAC) domain |
| Representative enzymes | DisA, CdaA, CdaS, ssDacA, Cas10-associated cyclases |
What Is GO:0106408?
In simple terms, diadenylate cyclase activity is the enzyme function that joins two ATP molecules into one cyclic di-AMP molecule while releasing two diphosphate groups. According to the QuickGO definition, it catalyzes the reaction 2 ATP = 3',3'-c-di-AMP + 2 diphosphate. The synonym cyclic-di-AMP synthase reflects this synthetic role. The activity resides in a conserved DisA_N (DAC) domain and is the only known route for c-di-AMP production in bacteria.
Why Is diadenylate cyclase activity Important in Cell Biology?
Diadenylate cyclase activity is important because it produces cyclic di-AMP, a second messenger that is essential or conditionally essential in many Gram-positive bacteria and that controls cell wall synthesis, potassium homeostasis, DNA damage responses and virulence. Because c-di-AMP is absent from human cells, the enzymes that make it are attractive targets for antimicrobial development, and inhibitors such as polyphenols and IPA-3 have been identified. Understanding how DAC activity is regulated by autoinhibitory domains, DNA intermediates and substrate pools informs both basic bacterial signalling biology and drug discovery.
• Produces the second messenger c-di-AMP, which is required for cell wall homeostasis and osmotic stress resistance in many bacteria.
• The founding DAC DisA couples c-di-AMP synthesis to DNA recombination intermediate surveillance, linking metabolism to genome integrity.
• DACs are widespread across bacterial and archaeal phyla with diverse domain architectures, making them a model for second messenger evolution.
• Autoinhibitory domains such as that of CdaS provide a paradigm for post-translational control of nucleotide cyclases.
• Inhibitors of DAC activity, including polyphenols and IPA-3, are chemical probes and potential antimicrobial leads.
• ssDacA in Streptococcus suis is a validated target for inhibitor development against this zoonotic pathogen.
• A miniature CRISPR-Cas10 enzyme uses an inhibitory signalling mechanism related to diadenylate cyclase activity, connecting this GO term to prokaryotic immunity.
• Dysregulation of c-di-AMP levels affects bacterial virulence and host immune activation, making DACs relevant to infection biology.
• DAC activity is a selectable and quantifiable enzymatic function suitable for high-throughput screening.
• Studying DACs informs synthetic biology approaches to control second messenger signalling in engineered bacteria.
Molecular Mechanism of diadenylate cyclase activity
Substrate binding and catalytic condensation
In simple terms: The enzyme grabs two ATP molecules and stitches them together into a ring.
Diadenylate cyclase activity catalyzes the condensation of two ATP molecules into one 3',3'-c-di-AMP molecule with release of two diphosphate groups. The reaction is carried out by the conserved DisA_N (DAC) domain, which binds ATP and coordinates the cyclization chemistry. Structural and biochemical work on DisA from Thermotoga maritima established the catalytic framework for this family.
Domain architecture and oligomeric assembly
In simple terms: Different DAC enzymes are built from different protein modules, but they all share the same catalytic core.
Diadenylate cyclases display diverse domain architectures in bacteria and archaea, but all contain the DisA_N (DAC) domain responsible for cyclase activity. Some DACs, such as DisA, assemble into oligomeric complexes that are important for regulation and for sensing DNA structures. The domain context determines whether the enzyme is a standalone cyclase or part of a larger signalling or defense protein.
Regulation by DNA recombination intermediates
In simple terms: Some DAC enzymes check DNA health before making the signal.
The DisA checkpoint protein binds DNA recombination intermediates, and this binding regulates its diadenylate cyclase activity. This couples c-di-AMP synthesis to the status of the chromosome, allowing the cell to respond to DNA damage or stalled replication. This regulatory logic is a hallmark of the founding member of the DAC family.
Autoinhibitory control of c-di-AMP production
In simple terms: Some DAC enzymes carry their own brake to avoid making too much signal.
CdaS in Bacillus subtilis contains an autoinhibitory domain that limits cyclic di-AMP production. This intramolecular control ensures that c-di-AMP levels are kept within a physiological range. Replenishing the c-di-AMP pool therefore depends on both enzyme abundance and relief of autoinhibition.
Chemical inhibition of DAC activity
In simple terms: Small molecules can jam the enzyme and stop the signal.
Polyphenols inhibit the diadenylate cyclase DisA, demonstrating that the active site is druggable. IPA-3 inhibits the diadenylate cyclase of Streptococcus suis and shows potent antimicrobial activity, linking enzyme inhibition to bacterial killing. These inhibitors are useful probes for dissecting c-di-AMP biology and for antimicrobial development.
DAC-related signalling in CRISPR-Cas10 immunity
In simple terms: A tiny CRISPR enzyme uses a DAC-like signal to defend bacteria.
A miniature CRISPR-Cas10 enzyme confers immunity by an inhibitory signalling mechanism that involves a diadenylate cyclase-related activity. This expands the biological roles of the DAC domain beyond classical second messenger production. It also highlights the evolutionary versatility of the DisA_N (DAC) fold.
Key Genes Involved in GO:0106408 diadenylate cyclase activity
The following genes and proteins are experimentally characterized representatives of diadenylate cyclase activity (GO:0106408) or are directly associated with its regulation and inhibition.
| Gene | Major Role | Research Relevance |
|---|---|---|
| disA (Thermotoga maritima) | Founding diadenylate cyclase; DNA recombination intermediate-binding checkpoint protein | Structural and biochemical model for DAC catalysis and DNA-regulated activity |
| cdaA (Bacillus subtilis) | Membrane-associated diadenylate cyclase producing c-di-AMP | Model for essential c-di-AMP synthesis and cell wall homeostasis |
| cdaS (Bacillus subtilis) | Diadenylate cyclase with an autoinhibitory domain | Paradigm for autoinhibitory control of c-di-AMP production |
| ssDacA (SSU98_1483, Streptococcus suis) | Diadenylate cyclase in Streptococcus suis serotype 2 | Validated target for antimicrobial inhibitor development |
| dacA (Streptococcus suis) | Diadenylate cyclase targeted by IPA-3 | Enzyme inhibition linked to potent antimicrobial activity |
| DisA_N (DAC) domain-containing genes | Conserved catalytic domain family across bacteria and archaea | Comparative genomics and domain architecture analysis |
| Cas10-associated cyclase genes | Miniature CRISPR-Cas10 immunity via inhibitory signalling | Links DAC activity to prokaryotic defense |
| cdaA homologs in Firmicutes | c-di-AMP synthesis and stress response | Genetics of second messenger pools |
| cdaS homologs in Bacillales | Spore-related c-di-AMP control | Developmental regulation of DAC activity |
| disA homologs in Thermotogae | DNA integrity checkpoint signalling | Coupling of DNA status to c-di-AMP |
| dacA homologs in Streptococcaceae | Virulence-associated c-di-AMP production | Pathogenesis and drug target studies |
| Polyphenol-sensitive DACs | Enzymes inhibited by polyphenols | Chemical biology of DAC inhibition |
| IPA-3-sensitive DACs | Enzymes inhibited by IPA-3 | Antimicrobial lead optimization |
| Membrane-associated DACs | Peripheral membrane cyclases | Membrane signalling and cell envelope stress |
| Soluble DACs | Cytoplasmic cyclases | Biochemical assay development |
| Oligomeric DACs | Assembly-dependent cyclases | Structure-function studies |
| Autoinhibited DACs | Enzymes with intrinsic regulatory domains | Allosteric regulation research |
| DAC-domain fusion proteins | Cyclase domains fused to sensory or defense modules | Domain architecture and evolution |
How Is diadenylate cyclase activity Regulated?
Diadenylate cyclase activity is regulated at multiple levels. The founding enzyme DisA is controlled by binding to DNA recombination intermediates, which modulates its cyclase output. CdaS from Bacillus subtilis is restrained by an autoinhibitory domain that limits cyclic di-AMP production. More broadly, the size of the c-di-AMP pool depends on the availability of ATP and on the balance between synthesis and degradation, a process reviewed as replenishing the cyclic-di-AMP pool. Chemical inhibitors such as polyphenols and IPA-3 provide external control of DAC activity and confirm that the active site is regulatable. Domain architecture also influences regulation, since DAC domains occur in diverse multidomain proteins that may integrate additional signals.
diadenylate cyclase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ssDacA (Streptococcus suis) | Streptococcus suis infection and virulence | Streptococcus suis knockout and inhibitor testing |
| dacA (Streptococcus suis) | Antimicrobial target | Enzyme inhibition assays with IPA-3 |
| cdaA (Bacillus subtilis) | Cell wall homeostasis and stress response | Bacillus subtilis knockout and c-di-AMP quantification |
| disA (Thermotoga maritima) | DNA damage checkpoint signalling | Recombinant DisA biochemical assays |
| cdaS (Bacillus subtilis) | Autoinhibitory control of c-di-AMP | Domain deletion and activity assays |
Bacterial infection and virulence
Diadenylate cyclase activity contributes to the production of c-di-AMP, a second messenger that supports bacterial growth, stress resistance and virulence. In Streptococcus suis, the diadenylate cyclase ssDacA is expressed and its activity is relevant to the biology of this zoonotic pathogen. Inhibiting this activity with IPA-3 has potent antimicrobial effects, suggesting that DAC enzymes are antibacterial drug targets.
Antimicrobial resistance and drug discovery
Because c-di-AMP signalling is absent in humans, diadenylate cyclases are attractive narrow-spectrum targets. Polyphenols inhibit DisA and provide chemical starting points for inhibitor development. IPA-3 inhibition of the Streptococcus suis DAC demonstrates proof of concept that blocking this activity can kill bacteria.
Host immune activation by c-di-AMP
Cyclic di-AMP produced by diadenylate cyclase activity is a potent immune stimulator, and its release during infection triggers host responses. The balance of c-di-AMP production therefore influences the inflammatory outcome of bacterial infection. Understanding DAC regulation helps explain how pathogens modulate host immunity.
Prokaryotic immunity and CRISPR-Cas10
A miniature CRISPR-Cas10 enzyme confers immunity through an inhibitory signalling mechanism involving a diadenylate cyclase-related activity. This connects GO:0106408 to the biology of CRISPR-based defense systems. It also suggests that DAC-like signalling modules can be repurposed for biotechnology applications.
From diadenylate cyclase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is the DAC gene essential for bacterial growth? | Knockout cell model in the target bacterium |
| Does a point mutation abolish catalytic activity? | Point-mutation knock-in of the DAC active site |
| How does an autoinhibitory domain control activity? | Domain-deletion knock-in and activity assays |
| Where is the DAC protein localized? | Tagged knock-in with fluorescent or affinity tag |
| Does overexpression increase c-di-AMP levels? | Overexpression cell model and c-di-AMP quantification |
| Can inhibitors block DAC activity in cells? | Wild-type and inhibitor-treated cell models |
How to Study the diadenylate cyclase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Recombinant DAC activity assay | Conversion of ATP to c-di-AMP | Enzyme kinetics and regulation |
| LC-MS/MS quantification | Cellular c-di-AMP levels | Knockout phenotyping |
| Inhibitor screening | Enzyme inhibition by small molecules | Antimicrobial lead discovery |
| Domain architecture analysis | Protein family classification | Comparative genomics of DACs |
| Site-directed mutagenesis | Catalytic residue requirement | Mechanistic studies |
| DNA-binding assays | Interaction with recombination intermediates | DisA regulation studies |
| Autoinhibition domain deletion | Relief of intrinsic inhibition | CdaS regulation studies |
| Antimicrobial susceptibility testing | Bacterial killing by DAC inhibitors | IPA-3 evaluation |
Enzymatic activity assays for diadenylate cyclase
Diadenylate cyclase activity is typically measured by incubating recombinant enzyme with ATP and detecting the formation of 3',3'-c-di-AMP, for example by chromatography or mass spectrometry. Such assays were used to characterize DisA and to show that DNA recombination intermediates regulate its activity. They are also used to evaluate inhibitors such as polyphenols and IPA-3.
Genetic knockouts and c-di-AMP quantification
Knocking out DAC genes and measuring cellular c-di-AMP levels reveals the contribution of each enzyme to the second messenger pool. This approach has been applied to cdaA and cdaS in Bacillus subtilis and to ssDacA in Streptococcus suis. Quantification can be performed by LC-MS/MS or by reporter systems responsive to c-di-AMP.
Structural and domain analysis
Structural studies of DisA revealed the fold of the DisA_N (DAC) domain and its DNA-binding regulatory module. Comparative domain architecture analysis across bacterial and archaeal genomes classifies DAC proteins into families. These methods guide mutagenesis and inhibitor design.
Inhibitor screening and chemical biology
High-throughput screening of small molecules against DAC enzymes identifies inhibitors such as polyphenols and IPA-3. Secondary assays confirm target engagement and antimicrobial activity. These methods support drug discovery aimed at c-di-AMP signalling.
How CRISPR Can Be Used to Study GO:0106408 diadenylate cyclase activity
Knockout
CRISPR knockout of diadenylate cyclase genes such as cdaA, cdaS or ssDacA allows researchers to determine their contribution to c-di-AMP pools and bacterial phenotypes. Knockout strains can be tested for growth, stress sensitivity and virulence. This approach is foundational for target validation in antimicrobial discovery.
Point Mutation
CRISPR point mutation of catalytic residues in the DisA_N (DAC) domain can separate cyclase activity from other functions of the protein. Such mutants help confirm that a observed phenotype depends on c-di-AMP synthesis. They are also useful for testing inhibitor specificity.
Knock-in
CRISPR knock-in of tagged or reporter-linked DAC genes enables localization and interaction studies. Knock-in of autoinhibition-domain variants can test regulatory models for CdaS. These models support structure-function analysis in the native genomic context.
Overexpression
CRISPR overexpression or inducible expression of DAC genes increases c-di-AMP production and reveals downstream signalling effects. Overexpression models are useful for biochemical purification and for testing inhibitor efficacy under high enzyme load. They also help define the upper limits of c-di-AMP tolerance in the cell.
How EDITGENE Supports diadenylate cyclase activity Research
Researchers studying diadenylate cyclase activity-related genes often need to determine whether a candidate gene is causally involved in c-di-AMP production, stress responses or virulence, and whether a specific catalytic residue or regulatory domain is required. EDITGENE provides the cell models and screening services needed to answer these questions with rigor and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for diadenylate cyclase activity research.
Frequently Asked Questions About diadenylate cyclase activity
What is diadenylate cyclase activity?
Diadenylate cyclase activity (GO:0106408) is the enzymatic activity that catalyzes the reaction 2 ATP = 3',3'-c-di-AMP + 2 diphosphate, producing the second messenger cyclic di-AMP.
What genes are involved in diadenylate cyclase activity?
Genes encoding the DisA_N (DAC) domain include disA, cdaA, cdaS and ssDacA, which are found across bacteria and archaea.
What is the reaction catalyzed by diadenylate cyclase?
The enzyme converts two ATP molecules into one 3',3'-c-di-AMP molecule and two diphosphate molecules.
Which enzyme is the founding member of the diadenylate cyclase family?
DisA from Thermotoga maritima was the first structurally and biochemically characterized diadenylate cyclase and is regulated by DNA recombination intermediates.
How is diadenylate cyclase activity regulated?
It is regulated by DNA binding in DisA, by an autoinhibitory domain in CdaS, and by the availability of ATP and the balance of c-di-AMP synthesis and degradation.
Can diadenylate cyclase activity be inhibited?
Yes, polyphenols inhibit DisA and IPA-3 inhibits the Streptococcus suis diadenylate cyclase with potent antimicrobial activity.
Why is diadenylate cyclase activity important for bacteria?
It produces c-di-AMP, which controls cell wall homeostasis, stress responses and virulence in many bacteria.
Is diadenylate cyclase activity a drug target?
Because c-di-AMP signalling is absent in humans, DAC enzymes are attractive antimicrobial targets, and inhibitors have shown proof of concept.
What is the role of the DisA_N domain?
The DisA_N (DAC) domain is the conserved catalytic module that carries out diadenylate cyclase activity in diverse protein architectures.
How do researchers study diadenylate cyclase activity?
They use recombinant enzyme assays, LC-MS/MS quantification of c-di-AMP, genetic knockouts, structural analysis and inhibitor screening.
Conclusion
Diadenylate cyclase activity (GO:0106408) is the conserved enzymatic function that produces the bacterial second messenger cyclic di-AMP from ATP. Its founding member DisA couples c-di-AMP synthesis to DNA integrity surveillance, while enzymes such as CdaS and ssDacA illustrate autoinhibitory and pathogen-specific regulation. Because c-di-AMP signalling is absent in humans, DAC enzymes are promising antimicrobial targets, and inhibitors such as polyphenols and IPA-3 validate this concept. Continued research using CRISPR knockouts, point mutations, knock-ins and overexpression models will clarify how this activity shapes bacterial physiology, immunity and infection.
References
- 1. Pham TH et al.. 2016. Replenishing the cyclic-di-AMP pool: regulation of diadenylate cyclase activity in bacteria.. Curr Genet 62(4):731-738 PMID: 27074767
- 2. Li H et al.. 2022. IPA-3: An Inhibitor of Diadenylate Cyclase of Streptococcus suis with Potent Antimicrobial Activity.. Antibiotics (Basel) 11(3) PMID: 35326881
- 3. Doherty EE et al.. 2025. A miniature CRISPR-Cas10 enzyme confers immunity by inhibitory signalling.. Nature 647(8091):997-1004 PMID: 41034576
- 4. Witte G et al.. 2008. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates.. Mol Cell 30(2):167-78 PMID: 18439896
- 5. Galperin MY. 2023. All DACs in a Row: Domain Architectures of Bacterial and Archaeal Diadenylate Cyclases.. J Bacteriol 205(4):e0002323 PMID: 37022175
- 6. Du B et al.. 2015. Diadenylate cyclase evaluation of ssDacA (SSU98_1483) in Streptococcus suis serotype 2.. Genet Mol Res 14(2):6917-24 PMID: 26125899
- 7. Mehne FM et al.. 2014. Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.. J Biol Chem 289(30):21098-107 PMID: 24939848
- 8. Opoku-Temeng C et al.. 2016. Inhibition of cyclic diadenylate cyclase, DisA, by polyphenols.. Sci Rep 6:25445 PMID: 27150552
