GO:0004639 phosphoribosylaminoimidazolesuccinocarboxamide synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004639 describes the enzymatic activity that catalyzes the ATP-dependent ligation of 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) with L-aspartate to form SAICAR, a key step in de novo purine biosynthesis.
• In humans, this activity is carried out by the bifunctional enzyme PAICS, which also possesses phosphoribosylaminoimidazole carboxylase activity.
• PAICS is overexpressed in multiple cancers and is considered an emerging cancer target.
• Structural studies of SAICAR synthase from bacteria and humans have revealed the catalytic mechanism and substrate binding.
• Deficiency or inhibition of this activity disrupts purine nucleotide pools, affecting cell proliferation and virulence in pathogens.
• CRISPR-based models (knockout, point mutation, knock-in, overexpression) enable precise interrogation of GO:0004639 in health and disease.
Description
Phosphoribosylaminoimidazolesuccinocarboxamide synthase activity (GO:0004639) is a molecular function that catalyzes the eighth step of the de novo purine biosynthesis pathway. This enzymatic activity, often referred to as SAICAR synthase or SAICAR synthetase, is responsible for the ATP-dependent ligation of CAIR and L-aspartate to produce SAICAR, ADP, phosphate, and protons. The reaction is essential for the production of purine nucleotides, which are required for DNA and RNA synthesis, energy metabolism, and cell signaling. In humans, this activity resides in the bifunctional enzyme PAICS, which also catalyzes the preceding step in the pathway. Because of its central role in nucleotide biosynthesis, SAICAR synthase activity has attracted attention as a potential therapeutic target in cancer and infectious diseases. Researchers studying this activity use a combination of structural biology, enzymology, and CRISPR-based genetic models to dissect its mechanism and cellular functions.
phosphoribosylaminoimidazolesuccinocarboxamide synthase activity At A Glance
| GO ID | GO:0004639 |
|---|---|
| GO term | phosphoribosylaminoimidazolesuccinocarboxamide synthase activity |
| Ontology | molecular_function |
| Synonym | SAICAR synthase activity; SAICAR synthetase activity; PurC; 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase activity |
| Major function | Catalyzes the ATP-dependent ligation of CAIR and L-aspartate to form SAICAR in de novo purine biosynthesis |
| Reaction | CAIR + L-aspartate + ATP = SAICAR + ADP + 2 H+ + phosphate |
| Pathway | De novo purine biosynthesis (IMP biosynthesis) |
| Human gene | PAICS (bifunctional phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase) |
| Bacterial gene | purC (e.g., Escherichia coli, Salmonella enterica, Xanthomonas campestris) |
What Is GO:0004639?
GO:0004639 is defined as the catalysis of the reaction: 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate + L-aspartate + ATP = (2S)-2-[5-amino-1-(5-phospho-beta-D-ribosyl)imidazole-4-carboxamido]succinate + ADP + 2 H+ + phosphate. In simpler terms, it is the enzyme activity that attaches aspartate to CAIR using ATP energy to form SAICAR, a precursor in purine biosynthesis. This activity is synonymous with SAICAR synthase, SAICAR synthetase, and PurC in bacteria.
Why Is phosphoribosylaminoimidazolesuccinocarboxamide synthase activity Important in Cell Biology?
Phosphoribosylaminoimidazolesuccinocarboxamide synthase activity is critical for de novo purine biosynthesis, which supplies the nucleotides needed for DNA replication, RNA transcription, and cellular energy metabolism. In humans, the bifunctional enzyme PAICS harboring this activity is overexpressed in several cancers and is linked to poor prognosis, making it an emerging target for anticancer therapy. In bacteria, the orthologous PurC enzyme is required for virulence and survival in host environments. Moreover, mutations or inhibition of this activity can lead to purine auxotrophy, providing a basis for selective antimicrobial and anticancer strategies.
• Essential for de novo purine biosynthesis, providing precursors for DNA and RNA.
• Human PAICS is overexpressed in cancers such as prostate cancer and is a potential therapeutic target.
• Bacterial PurC is required for virulence in pathogens like Xanthomonas campestris.
• Deficiency in purine de novo synthesis affects cell proliferation and metabolism.
• SAICAR, the product, has been implicated in regulation of thiamine synthesis in Salmonella.
• Structural knowledge of SAICAR synthase enables rational drug design.
• CRISPR screens can identify dependencies on this activity in cancer cells.
• Enzyme assays and metabolomics can measure pathway flux and guide inhibitor development.
Molecular Mechanism of phosphoribosylaminoimidazolesuccinocarboxamide synthase activity
Substrate Binding and Catalysis
In simple terms: The enzyme grabs CAIR and aspartate, then uses ATP to join them together.
SAICAR synthase binds the substrate CAIR and L-aspartate in a sequential manner. Structural studies of bacterial and human enzymes have revealed that ATP binds first, followed by CAIR and aspartate, leading to the formation of a ternary complex. The reaction proceeds via an acyl phosphate intermediate, where the carboxylate of CAIR is activated by ATP to form a mixed anhydride, which is then attacked by the amino group of aspartate to yield SAICAR, ADP, and phosphate.
Role of Divalent Cations
In simple terms: Magnesium ions help ATP do its job.
The catalytic activity requires divalent cations, typically Mg2+, which coordinate the phosphate groups of ATP and stabilize the transition state. Crystallographic studies of E. coli PurC in complex with nucleotides showed that Mg2+ is essential for ATP binding and catalysis.
Bifunctional Enzyme in Humans
In simple terms: In humans, the same protein does two steps in a row.
Human PAICS is a bifunctional enzyme that catalyzes both the carboxylation of AIR to CAIR (EC 4.1.1.21) and the subsequent SAICAR synthase reaction (EC 6.3.2.6). The crystal structure of human PAICS revealed a decameric assembly with active sites for both activities, allowing efficient channeling of the intermediate CAIR. This bifunctional arrangement is also observed in other organisms.
Structural Insights and Catalytic Residues
In simple terms: The 3D shape of the enzyme shows exactly how it works.
The structure of SAICAR synthase from Thermus thermophilus and E. coli revealed a conserved fold with a central beta-sheet flanked by alpha-helices. Key catalytic residues include a conserved aspartate and a lysine that stabilize the substrate and transition state. Mutagenesis studies confirmed their essential roles in catalysis.
Regulation of Enzyme Activity
In simple terms: The cell controls how much of this enzyme is made and how active it is.
Expression of PAICS is regulated at the transcriptional level by factors such as MYC and E2F, which drive proliferation-associated metabolic programs. Additionally, feedback inhibition by purine nucleotides may modulate flux through the pathway. In bacteria, PurC expression is controlled by purine repressors.
Key Genes Involved in GO:0004639 phosphoribosylaminoimidazolesuccinocarboxamide synthase activity
The following genes encode enzymes with phosphoribosylaminoimidazolesuccinocarboxamide synthase activity or are directly related to its function across species.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PAICS (human) | Bifunctional enzyme with SAICAR synthase and AIR carboxylase activities | Overexpressed in cancers; target for inhibitor development |
| purC (E. coli) | Monofunctional SAICAR synthase | Model for structural and mechanistic studies |
| purC (S. enterica) | SAICAR synthase in purine biosynthesis | Linked to thiamine synthesis regulation |
| purC (X. campestris) | SAICAR synthase required for virulence | Plant pathogen model for purine auxotrophy |
| ADE1 (yeast) | Bifunctional SAICAR synthase | Eukaryotic model for purine pathway |
| PAICS (mouse) | Bifunctional enzyme | Knockout models for developmental studies |
| purC (B. subtilis) | SAICAR synthase | Gram-positive model for purine biosynthesis |
| purC (M. tuberculosis) | SAICAR synthase | Potential drug target in tuberculosis |
| PAICS (zebrafish) | Bifunctional enzyme | Developmental model for purine synthesis |
| purC (A. thaliana) | SAICAR synthase | Plant purine biosynthesis |
| PAICS (D. melanogaster) | Bifunctional enzyme | Genetic model for purine disorders |
| purC (C. elegans) | SAICAR synthase | Model for purine metabolism |
| PAICS (rat) | Bifunctional enzyme | Biochemical studies |
| purC (S. cerevisiae) | SAICAR synthase | Yeast genetics |
| PAICS (chicken) | Bifunctional enzyme | Developmental biology |
| purC (V. cholerae) | SAICAR synthase | Pathogenesis studies |
How Is phosphoribosylaminoimidazolesuccinocarboxamide synthase activity Regulated?
The expression and activity of phosphoribosylaminoimidazolesuccinocarboxamide synthase are regulated at multiple levels. In humans, the PAICS gene is a direct target of the MYC oncogene, which drives its overexpression in many cancers. The promoter region contains E2F binding sites, linking its expression to the cell cycle. In bacteria, PurC is part of the pur operon, which is repressed by purine nucleotides via the PurR repressor. Additionally, feedback inhibition by downstream purine nucleotides may regulate enzymatic activity. Post-translational modifications, such as phosphorylation, have been detected on PAICS but their functional impact requires further study.
phosphoribosylaminoimidazolesuccinocarboxamide synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PAICS | Cancer (prostate, lung, glioblastoma) | PAICS knockout or overexpression in cancer cell lines |
| purC (X. campestris) | Bacterial virulence | purC deletion mutant in plant infection model |
| purC (S. enterica) | Thiamine synthesis regulation | purC mutant for metabolic profiling |
| PAICS | Purine metabolism disorders | Patient-derived fibroblasts with PAICS mutations |
| purC (M. tuberculosis) | Tuberculosis | purC knockout in M. tuberculosis |
Cancer
PAICS, the human enzyme with SAICAR synthase activity, is overexpressed in various malignancies, including prostate cancer, lung cancer, and glioblastoma. High PAICS expression correlates with poor prognosis and promotes cell proliferation by sustaining purine nucleotide pools. Knockdown of PAICS inhibits tumor growth in xenograft models, validating it as a potential therapeutic target.
Infectious Diseases
In bacterial pathogens such as Xanthomonas campestris and Salmonella enterica, PurC is essential for virulence and survival within the host. Mutants lacking purC are auxotrophic for purines and exhibit reduced virulence in plant and animal models. Thus, SAICAR synthase is a candidate target for antimicrobial development.
Inborn Errors of Metabolism
Defects in purine de novo synthesis can lead to severe metabolic disorders, including immunodeficiency and neurological abnormalities. Although specific mutations in PAICS have not been widely reported, deficiencies in upstream enzymes cause SAICAR accumulation, which may be toxic.
From phosphoribosylaminoimidazolesuccinocarboxamide synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PAICS loss inhibit tumor growth? | PAICS knockout in cancer cell lines and xenografts |
| What is the catalytic mechanism of SAICAR synthase? | Point mutations in catalytic residues of bacterial PurC |
| How does SAICAR synthase deficiency affect metabolism? | Knockout of purC in S. enterica followed by metabolomics |
| Can PAICS be targeted by inhibitors? | Knock-in of tagged PAICS for drug binding assays |
| What is the role of PAICS in development? | Conditional knockout in mouse models |
| How does PAICS overexpression affect cell proliferation? | Overexpression of PAICS in normal cells |
How to Study the phosphoribosylaminoimidazolesuccinocarboxamide synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | SAICAR synthase activity | Inhibitor screening, mutant characterization |
| X-ray crystallography | 3D structure of enzyme-substrate complexes | Mechanistic studies, drug design |
| Metabolomics | Levels of purine intermediates | Pathway flux analysis |
| CRISPR knockout screen | Gene essentiality | Identifying cancer dependencies |
| Western blot | Protein expression | Validating knockout or overexpression |
| qRT-PCR | mRNA levels | Gene expression analysis |
| Immunofluorescence | Subcellular localization | Studying enzyme localization |
| Enzyme kinetics | Km, Vmax, kcat | Characterizing mutants |
Enzymatic Assays
Direct measurement of SAICAR synthase activity can be performed using coupled spectrophotometric assays that monitor ADP formation or SAICAR production. Radioactive or fluorescent substrates allow sensitive detection. These assays are used to screen inhibitors and characterize mutants.
Structural Biology
X-ray crystallography and cryo-EM have been used to solve structures of SAICAR synthase from bacteria and humans, revealing substrate binding and catalytic mechanisms. These methods guide rational drug design.
Metabolomics
Targeted and untargeted metabolomics can quantify purine intermediates, including SAICAR, in cells with altered SAICAR synthase activity. This approach has been applied to HeLa cells deficient in purine de novo synthesis.
CRISPR Screens
Genome-wide CRISPR knockout screens can identify cancer cell lines that are dependent on PAICS for proliferation. Such screens have highlighted PAICS as a selective dependency in certain cancers.
How CRISPR Can Be Used to Study GO:0004639 phosphoribosylaminoimidazolesuccinocarboxamide synthase activity
Knockout
CRISPR-Cas9 knockout of PAICS or purC eliminates SAICAR synthase activity, leading to purine auxotrophy and growth arrest in cells that rely on de novo synthesis. Knockout models are used to study metabolic dependencies and validate drug targets.
Point Mutation
Introducing point mutations in catalytic residues (e.g., aspartate or lysine) of SAICAR synthase via CRISPR base editing or homology-directed repair allows precise dissection of the catalytic mechanism and substrate specificity.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins at the endogenous PAICS locus enables real-time imaging, immunoprecipitation, and proteomic analysis of the enzyme complex.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of PAICS can model the overexpression observed in cancers, allowing researchers to study its oncogenic effects and test targeted therapies.
How EDITGENE Supports phosphoribosylaminoimidazolesuccinocarboxamide synthase activity Research
Researchers studying phosphoribosylaminoimidazolesuccinocarboxamide synthase activity-related genes often need to determine whether a candidate gene is causally involved in purine metabolism, cancer progression, or microbial virulence. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models, enabling functional validation and drug discovery.
Contact EDITGENE today to design your custom CRISPR model for phosphoribosylaminoimidazolesuccinocarboxamide synthase activity research.
Frequently Asked Questions About phosphoribosylaminoimidazolesuccinocarboxamide synthase activity
What is phosphoribosylaminoimidazolesuccinocarboxamide synthase activity?
It is an enzymatic activity (GO:0004639) that catalyzes the ATP-dependent formation of SAICAR from CAIR and aspartate in de novo purine biosynthesis.
What genes are involved in phosphoribosylaminoimidazolesuccinocarboxamide synthase activity?
In humans, the PAICS gene encodes the bifunctional enzyme with this activity. In bacteria, the purC gene encodes the monofunctional enzyme.
What is the role of PAICS in cancer?
PAICS is overexpressed in several cancers and supports proliferation by maintaining purine nucleotide pools; it is considered a potential therapeutic target.
How is SAICAR synthase activity measured?
It can be measured using coupled enzymatic assays that detect ADP or SAICAR formation, often with purified recombinant enzyme or cell lysates.
What diseases are associated with defects in this activity?
Defects in purine de novo synthesis can cause metabolic disorders; in pathogens, loss of PurC reduces virulence.
What is the reaction catalyzed by SAICAR synthase?
CAIR + L-aspartate + ATP = SAICAR + ADP + 2 H+ + phosphate.
Can CRISPR be used to study phosphoribosylaminoimidazolesuccinocarboxamide synthase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models allow precise functional studies of this activity.
What is the difference between PAICS and PurC?
PAICS is a bifunctional human enzyme with both AIR carboxylase and SAICAR synthase activities, while PurC is a monofunctional bacterial SAICAR synthase.
Is SAICAR synthase a drug target?
Yes, its essential role in purine biosynthesis and overexpression in cancer make it an attractive target for inhibitor development.
How does SAICAR synthase deficiency affect cells?
Deficiency leads to purine auxotrophy, growth arrest, and in pathogens, reduced virulence.
Conclusion
Phosphoribosylaminoimidazolesuccinocarboxamide synthase activity (GO:0004639) is a fundamental enzymatic step in de novo purine biosynthesis, with critical roles in human health and disease. The bifunctional human enzyme PAICS and its bacterial counterpart PurC have been extensively characterized structurally and functionally, revealing druggable pockets and regulatory mechanisms. CRISPR-based models are invaluable for dissecting the precise contributions of this activity to cancer, infection, and metabolism. EDITGENE's comprehensive services empower researchers to create tailored cell models and accelerate discoveries targeting this pathway.
References
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- 5. Levdikov VM et al.. 1998. The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.. Structure 6(3):363-76 PMID: 9551557
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- 7. Yuan Z et al.. 2013. Genetic and proteomic analyses of a Xanthomonas campestris pv. campestris purC mutant deficient in purine biosynthesis and virulence.. J Genet Genomics 40(9):473-87 PMID: 24053949
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