GO:0003877 ATP:ADP adenylyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0003877 (ATP:ADP adenylyltransferase activity) catalyzes the reversible formation of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) from ATP and ADP, releasing phosphate.
• The reaction is a phosphorolysis that interconverts diadenosine tetraphosphate and adenine nucleotides, linking nucleotide pools to cellular energy charge.
• Enzymes with this activity belong to the Nudix hydrolase and histidine triad (HIT) families, and some are bifunctional with APS kinase or sulfurylase domains [1,6].
• Ap4A, the product of this activity, is a signaling molecule implicated in the cellular stress response, DNA repair, and metabolic regulation.
• Dysregulation of Ap4A metabolism has been linked to cancer, neurodegeneration, and metabolic disorders, making this activity a potential therapeutic target.
• CRISPR knockout, point mutation, and knock-in models are essential to dissect the physiological roles of ATP:ADP adenylyltransferase activity in vivo.
Description
ATP:ADP adenylyltransferase activity (GO:0003877) is a molecular function that catalyzes the reversible phosphorolysis of ATP and ADP to form P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and phosphate. This reaction is a key node in dinucleoside polyphosphate metabolism, influencing the cellular levels of Ap4A, a molecule that has been implicated in diverse processes ranging from stress responses to DNA repair. The activity is found in organisms across all domains of life, from bacteria to humans, and is often associated with bifunctional enzymes that also possess APS kinase or sulfurylase activities [1,6]. Researchers study GO:0003877 because it sits at the intersection of nucleotide metabolism and cellular signaling. The product Ap4A can act as an alarmone, modulating protein function and gene expression under conditions of cellular stress. Moreover, the enzyme catalyzing this reaction is a potential drug target, as its dysregulation has been observed in cancer and other diseases. Understanding the precise catalytic mechanism, regulation, and physiological roles of ATP:ADP adenylyltransferase activity requires a combination of biochemical, structural, and genetic approaches [1,6]. This article provides a comprehensive overview of GO:0003877, covering its definition, catalytic mechanism, key genes, disease associations, and the research methods used to study it. By integrating authoritative QuickGO data with real PubMed literature, we aim to equip researchers with a solid foundation for investigating this important molecular function.
ATP:ADP adenylyltransferase activity At A Glance
| GO ID | GO:0003877 |
|---|---|
| GO term | ATP:ADP adenylyltransferase activity |
| Ontology | molecular_function |
| Synonym | diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase activity; AP-4-A phosphorylase activity; ATP adenylyltransferase activity |
| Major function | Catalyzes the reversible formation of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) from ATP and ADP, releasing phosphate |
| Reaction | ADP + ATP = phosphate + P(1),P(4)-bis(5'-adenosyl)tetraphosphate |
| EC number | 2.7.7.53 (not explicitly in QuickGO but commonly associated) |
| Subcellular location | Cytosol, nucleus, and mitochondria (varies by organism) |
| Related pathways | Nucleotide metabolism, stress response, DNA repair |
What Is GO:0003877?
ATP:ADP adenylyltransferase activity (GO:0003877) is defined as the catalysis of the reaction: ADP + ATP = phosphate + P(1),P(4)-bis(5'-adenosyl)tetraphosphate. In other words, it is an enzyme activity that transfers an adenylyl group from ATP to ADP, forming a dinucleoside tetraphosphate (Ap4A) and releasing inorganic phosphate. This reaction is reversible and represents a phosphorolytic cleavage of Ap4A. The activity is also known by several synonyms, including diadenosine tetraphosphate phosphorylase, AP-4-A phosphorylase, and bis(5'-nucleosyl)-tetraphosphate phosphorylase (NDP-forming).
Why Is ATP:ADP adenylyltransferase activity Important in Cell Biology?
ATP:ADP adenylyltransferase activity is important because it regulates the cellular levels of Ap4A, a dinucleoside polyphosphate that functions as a signaling molecule in stress responses, DNA repair, and metabolic regulation. By controlling Ap4A homeostasis, this activity influences fundamental cellular processes such as proliferation, differentiation, and apoptosis. Furthermore, the enzyme catalyzing this reaction is a potential therapeutic target, as its dysregulation has been linked to cancer, neurodegeneration, and metabolic disorders. Understanding this activity is therefore crucial for both basic biology and translational research.
• Regulates Ap4A levels, which act as alarmones under stress conditions.
• Influences nucleotide pool balance and energy charge.
• Implicated in DNA repair and genomic stability.
• Linked to cancer cell proliferation and survival.
• Potential target for anti-cancer and anti-neurodegenerative therapies.
• Plays a role in bacterial stress response and virulence.
• Involved in plant development and photosynthetic metabolism.
• Associated with metabolic disorders such as diabetes.
• Provides a model for studying enzyme evolution and bifunctional catalysis [1,6].
• Enables research on dinucleoside polyphosphate signaling.
What Happens During ATP:ADP adenylyltransferase activity?
Substrate Binding and Catalysis
In simple terms: The enzyme grabs ATP and ADP and joins them together, releasing a phosphate.
The catalytic cycle begins with the binding of ATP and ADP to the active site of the enzyme. The enzyme facilitates the transfer of an adenylyl group from ATP to ADP, forming P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and inorganic phosphate. This reaction is reversible, and the enzyme can also catalyze the reverse phosphorolysis of Ap4A to generate ATP and ADP. Structural studies of related enzymes, such as the bifunctional APS kinase domain of human PAPS synthetase 1, have revealed key active-site residues involved in nucleotide binding and catalysis.
Role of Metal Ions and Cofactors
In simple terms: Metal ions like magnesium help the enzyme work properly.
Many adenylyltransferases require divalent metal ions, such as Mg2+ or Mn2+, for catalytic activity. These ions stabilize the phosphate groups of ATP and ADP and facilitate the nucleophilic attack during the transfer reaction. In some enzymes, the metal ion is coordinated by conserved aspartate or histidine residues in the active site. The exact metal requirements can vary among different ATP:ADP adenylyltransferases, and some enzymes may function without metal ions.
Bifunctional Enzymes and Domain Organization
In simple terms: Some enzymes have two jobs, like a Swiss Army knife.
Several enzymes that exhibit ATP:ADP adenylyltransferase activity are bifunctional, containing additional domains such as APS kinase or ATP sulfurylase [1,6]. For example, the human PAPS synthetase 1 has an APS kinase domain that catalyzes the phosphorylation of APS to PAPS, and this domain also displays adenylyltransferase activity. The domain organization allows for coordinated regulation of nucleotide metabolism and sulfate activation. The crystal structure of phosphopantetheine adenylyltransferase from Enterococcus faecalis in complex with ATP and pantetheine provides insights into how these enzymes bind nucleotides and catalyze adenylyl transfer.
Regulation by Cellular Energy Charge
In simple terms: The enzyme senses the cell's energy level and adjusts its activity.
The activity of ATP:ADP adenylyltransferase is sensitive to the cellular energy charge, as its substrates (ATP and ADP) are key energy carriers. Changes in the ATP/ADP ratio can directly affect the rate of Ap4A synthesis. In Escherichia coli, the cellular levels of glucose 6-phosphate and fructose 1,6-diphosphate, which reflect energy status, correlate with the rates of glycogen synthesis and glucose utilization, suggesting a link between energy charge and nucleotide metabolism. This regulation ensures that Ap4A production is coupled to the metabolic state of the cell.
Subcellular Localization and Compartmentalization
In simple terms: The enzyme can be found in different parts of the cell, affecting its function.
ATP:ADP adenylyltransferase activity has been detected in various subcellular compartments, including the cytosol, nucleus, and mitochondria. The localization can influence substrate availability and the downstream effects of Ap4A. For instance, nuclear Ap4A may participate in DNA repair and replication, while mitochondrial Ap4A could modulate oxidative phosphorylation. The plastidial localization of related enzymes in plants, such as ADP-glucose pyrophosphatase, highlights the importance of compartmentalization in metabolic regulation.
Key Genes Involved in GO:0003877 ATP:ADP adenylyltransferase activity
The following genes encode enzymes with ATP:ADP adenylyltransferase activity or are closely related to its function, based on published literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NUDT2 | Nudix hydrolase that hydrolyzes Ap4A; may also catalyze reverse reaction | Regulates Ap4A levels; implicated in cancer and stress response |
| NUDT4 | Nudix hydrolase with specificity for dinucleoside polyphosphates | Modulates Ap4A signaling; potential tumor suppressor |
| NUDT5 | Nudix hydrolase that hydrolyzes ADP-ribose and Ap4A | Involved in DNA repair and metabolic regulation |
| NUDT9 | ADP-ribose pyrophosphatase; may act on Ap4A | Linked to oxidative stress and neurodegeneration |
| HINT1 | Histidine triad nucleotide-binding protein; hydrolyzes Ap4A | Tumor suppressor; mutations in neuropathy |
| HINT2 | Mitochondrial HIT protein; Ap4A hydrolase | Regulates apoptosis and mitochondrial function |
| HINT3 | HIT family hydrolase; Ap4A metabolism | Poorly characterized; potential role in cancer |
| FHIT | Fragile histidine triad protein; hydrolyzes Ap4A | Frequently deleted in cancers; tumor suppressor |
| PAPSS1 | Bifunctional APS kinase and adenylyltransferase | Sulfate activation; cancer and developmental disorders |
| PAPSS2 | Bifunctional APS kinase and adenylyltransferase | Sulfate activation; skeletal dysplasia |
| ATP6V1E1 | V-ATPase subunit; not directly adenylyltransferase | Energy metabolism; cancer |
| GALK1 | Galactokinase; related to nucleotide metabolism | Galactosemia; metabolic disorders |
| ADK | Adenosine kinase; regulates adenosine levels | Neurological disorders; cancer |
| NME1 | Nucleoside diphosphate kinase; affects Ap4A levels | Metastasis suppressor |
| NME2 | Nucleoside diphosphate kinase; affects Ap4A levels | Cancer progression |
| AK1 | Adenylate kinase; energy homeostasis | Metabolic disorders; hemolytic anemia |
| AK2 | Adenylate kinase; energy homeostasis | Reticular dysgenesis |
| ENPP1 | Ectonucleotide pyrophosphatase; hydrolyzes Ap4A | Mineralization disorders; cancer |
How Is ATP:ADP adenylyltransferase activity Regulated?
The activity of ATP:ADP adenylyltransferase is regulated at multiple levels. Cellular energy charge directly influences substrate availability, as ATP and ADP levels fluctuate with metabolic state. In plants, thioredoxin f1 and NADPH-dependent thioredoxin reductase C regulate photosynthetic metabolism, which may indirectly affect nucleotide pools and adenylyltransferase activity. Additionally, the expression of genes encoding these enzymes can be modulated by stress-responsive transcription factors, such as those activated during oxidative stress or DNA damage. Post-translational modifications, including phosphorylation, may also regulate enzyme activity, although specific sites remain to be fully characterized.
ATP:ADP adenylyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| FHIT | Cancer (lung, breast, gastric) | Knockout mouse models; cancer cell lines |
| HINT1 | Axonal neuropathy with neuromyotonia | HINT1 knockout mice; patient-derived iPSCs |
| NUDT2 | Cancer; stress response | NUDT2 knockout cell lines; xenograft models |
| ENPP1 | Insulin resistance; ectopic mineralization | ENPP1 knockout mice; metabolic assays |
| PAPSS2 | Skeletal dysplasia; sulfate metabolism | PAPSS2 knockout zebrafish; chondrocyte cultures |
Cancer
Dysregulation of Ap4A metabolism has been observed in various cancers. The fragile histidine triad (FHIT) gene, which encodes an Ap4A hydrolase, is frequently deleted in many human cancers, including lung, breast, and gastric cancers. Loss of FHIT leads to elevated Ap4A levels, which can promote cell proliferation and survival. Similarly, NUDT2 and other Nudix hydrolases that regulate Ap4A are often altered in tumors, suggesting that ATP:ADP adenylyltransferase activity and Ap4A homeostasis are critical for cancer development.
Neurodegeneration
Ap4A has been implicated in neurodegenerative processes. Mutations in HINT1, a histidine triad hydrolase that metabolizes Ap4A, cause autosomal recessive axonal neuropathy with neuromyotonia. This suggests that proper regulation of Ap4A levels is essential for neuronal function. Additionally, oxidative stress in neurons can lead to increased Ap4A production, which may contribute to protein aggregation and cell death.
Metabolic Disorders
Alterations in nucleotide metabolism, including Ap4A levels, have been linked to metabolic disorders such as diabetes and obesity. For example, ENPP1, which hydrolyzes Ap4A, is associated with insulin resistance and ectopic mineralization. Furthermore, the activity of ATP:ADP adenylyltransferase may influence energy homeostasis through its effects on ATP and ADP pools, making it a potential target for metabolic disease therapies.
From ATP:ADP adenylyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the catalytic mechanism of ATP:ADP adenylyltransferase? | Recombinant enzyme with point mutations in active site; crystallography |
| How does loss of ATP:ADP adenylyltransferase affect Ap4A levels? | CRISPR knockout cell lines; mass spectrometry |
| What are the physiological roles of Ap4A in stress response? | Knockout mouse models; stress induction assays |
| How does ATP:ADP adenylyltransferase contribute to cancer? | Overexpression and knockout cancer cell lines; xenografts |
| What is the subcellular localization of the enzyme? | Tagged knock-in with fluorescent proteins; live-cell imaging |
| Can ATP:ADP adenylyltransferase be targeted therapeutically? | Small molecule screening; patient-derived organoids |
How to Study the ATP:ADP adenylyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC | Nucleotide and Ap4A levels | Enzyme activity assays; kinetic studies |
| Mass spectrometry | Ap4A and metabolite quantification | Metabolomics; flux analysis |
| X-ray crystallography | Three-dimensional protein structure | Active site mapping; inhibitor design [4,6] |
| CRISPR knockout | Gene function loss | Phenotypic analysis; Ap4A level changes |
| RNA-seq | Transcriptome changes | Pathway analysis upon gene perturbation |
| Proteomics | Protein expression and modifications | Identifying interacting partners; signaling |
| Live-cell imaging | Subcellular localization | Tagged knock-in; dynamics |
| CRISPR library screening | Genetic interactions | Synthetic lethality; drug resistance |
Biochemical Assays for Adenylyltransferase Activity
Enzymatic activity of ATP:ADP adenylyltransferase can be measured using high-performance liquid chromatography (HPLC) to separate and quantify nucleotides and Ap4A. Typically, recombinant enzyme is incubated with ATP and ADP, and the formation of Ap4A is monitored by UV absorbance or mass spectrometry. Radioactive labeling with 32P-ATP can also be used for sensitive detection.
Structural Biology Approaches
X-ray crystallography and cryo-electron microscopy can elucidate the three-dimensional structure of ATP:ADP adenylyltransferases in complex with substrates or inhibitors. For example, the crystal structure of phosphopantetheine adenylyltransferase from Enterococcus faecalis in complex with ATP and pantetheine revealed key active-site interactions. These studies inform rational drug design and mechanistic understanding.
Genetic and Genomic Methods
CRISPR-Cas9 knockout, point mutation, and knock-in strategies enable the study of gene function in cells and animal models. RNA-seq and proteomics can reveal global changes in gene expression and protein abundance upon perturbation of ATP:ADP adenylyltransferase activity. Additionally, CRISPR library screening can identify synthetic lethal interactions and pathways that buffer against loss of this activity.
Metabolomics and Flux Analysis
Mass spectrometry-based metabolomics allows quantification of Ap4A and related nucleotides in biological samples. Stable isotope tracing can measure flux through the adenylyltransferase reaction, providing insights into its contribution to nucleotide metabolism. These methods are essential for understanding how the activity affects cellular energy charge and signaling.
How CRISPR Can Be Used to Study GO:0003877 ATP:ADP adenylyltransferase activity
Knockout
CRISPR-Cas9 knockout of genes encoding ATP:ADP adenylyltransferase (e.g., NUDT2, FHIT) can abolish enzyme activity, leading to elevated Ap4A levels. These models are used to study the physiological consequences of Ap4A accumulation, such as altered stress responses, DNA repair defects, and cancer predisposition. Knockout cell lines and mouse models are valuable for validating the role of the enzyme in disease.
Point Mutation
Introducing point mutations in the catalytic residues of ATP:ADP adenylyltransferase can dissect the contribution of the enzymatic activity to its biological functions. For example, mutating the active-site histidine in HINT1 eliminates hydrolase activity and can mimic disease-causing mutations found in patients with neuropathy. Such models help distinguish between catalytic and non-catalytic roles of the protein.
Knock-in
Knock-in of tagged versions of the enzyme (e.g., GFP or FLAG) allows for real-time tracking of localization and interaction partners. Additionally, knock-in of disease-associated mutations (e.g., in HINT1 or PAPSS2) can create isogenic cell lines and animal models that recapitulate human pathology [1,6]. These models are essential for drug testing and mechanistic studies.
Overexpression
Overexpression of ATP:ADP adenylyltransferase or its mutant forms can lead to increased Ap4A levels and activation of downstream signaling pathways. Overexpression models are used to study the oncogenic potential of the enzyme and to screen for inhibitors that block its activity. They also help identify dose-dependent effects on cellular metabolism and proliferation.
How EDITGENE Supports ATP:ADP adenylyltransferase activity Research
Researchers studying ATP:ADP adenylyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in a specific phenotype, such as cancer cell proliferation or stress resistance. This requires precise genetic manipulation, which can be achieved through CRISPR-based knockout, point mutation, knock-in, or overexpression models. EDITGENE provides end-to-end services to generate these models and to perform downstream screening and bioinformatics analysis.
Contact EDITGENE today to design your custom CRISPR model for ATP:ADP adenylyltransferase activity research.
Frequently Asked Questions About ATP:ADP adenylyltransferase activity
What is ATP:ADP adenylyltransferase activity?
ATP:ADP adenylyltransferase activity (GO:0003877) is an enzyme activity that catalyzes the reversible formation of P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) from ATP and ADP, releasing phosphate.
What genes are involved in ATP:ADP adenylyltransferase activity?
Genes encoding enzymes with this activity include NUDT2, NUDT4, NUDT5, HINT1, HINT2, FHIT, and PAPSS1, among others [1,6].
What is the function of ATP:ADP adenylyltransferase?
It regulates the cellular levels of Ap4A, a signaling molecule involved in stress responses, DNA repair, and metabolic regulation.
How is ATP:ADP adenylyltransferase activity measured?
It can be measured using HPLC or mass spectrometry to quantify Ap4A formation from ATP and ADP, or by radioactive assays.
What diseases are associated with ATP:ADP adenylyltransferase dysfunction?
Dysregulation has been linked to cancer, neurodegenerative disorders such as HINT1-related neuropathy, and metabolic disorders.
What is the reaction catalyzed by ATP:ADP adenylyltransferase?
The reaction is: ADP + ATP = phosphate + P(1),P(4)-bis(5'-adenosyl)tetraphosphate.
What are the synonyms for ATP:ADP adenylyltransferase activity?
Synonyms include diadenosine tetraphosphate phosphorylase, AP-4-A phosphorylase, and bis(5'-nucleosyl)-tetraphosphate phosphorylase (NDP-forming).
How can CRISPR be used to study ATP:ADP adenylyltransferase activity?
CRISPR knockout, point mutation, knock-in, and overexpression models allow researchers to dissect the physiological roles of the enzyme and its product Ap4A.
What is the role of Ap4A in the cell?
Ap4A acts as an alarmone, modulating protein function and gene expression under stress conditions, and is involved in DNA repair and proliferation.
Which model organisms are used to study ATP:ADP adenylyltransferase?
Common models include Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and mammalian cell lines and mice [1,7].
Conclusion
ATP:ADP adenylyltransferase activity (GO:0003877) is a critical molecular function that governs the balance of diadenosine tetraphosphate (Ap4A) and adenine nucleotides. Its role in stress response, DNA repair, and metabolic regulation makes it a compelling target for both basic and translational research. Dysregulation of this activity has been implicated in cancer, neurodegeneration, and metabolic disorders, underscoring its clinical relevance. Advances in CRISPR-based genome editing, structural biology, and metabolomics are providing new tools to dissect the mechanism and physiological functions of ATP:ADP adenylyltransferase. By leveraging these approaches, researchers can uncover novel therapeutic strategies targeting this activity and its associated pathways [1,6].
References
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- 2. Mina M et al.. 1988. Assay of ATP-sulfurylase activity from rat liver by high-performance liquid chromatography.. J Chromatogr 433:63-72 PMID: 2853170
- 4. Yoon HJ et al.. 2011. Crystal structure of phosphopantetheine adenylyltransferase from Enterococcus faecalis in the ligand-unbound state and in complex with ATP and pantetheine.. Mol Cells 32(5):431-5 PMID: 21912874
- 5. Rodriguez-López M et al.. 2000. Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis.. Proc Natl Acad Sci U S A 97(15):8705-10 PMID: 10890880
- 6. Sekulic N et al.. 2007. Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1.. J Mol Biol 367(2):488-500 PMID: 17276460
- 7. Thormählen I et al.. 2015. Thioredoxin f1 and NADPH-Dependent Thioredoxin Reductase C Have Overlapping Functions in Regulating Photosynthetic Metabolism and Plant Growth in Response to Varying Light Conditions.. Plant Physiol 169(3):1766-86 PMID: 26338951
- 8. Dietzler DN et al.. 1979. Regulation of glycogen synthesis and glucose utilization in Escherichia coli during maintenance of the energy charge. Quantitative correlation of changes in the rates of glycogen synthesis and glucose utilization with simultaneous changes in the cellular levels of both glucose 6-phosphate and fructose 1,6-diphosphate.. J Biol Chem 254(17):8276-87 PMID: 381301
