GO:0004642 phosphoribosylformylglycinamidine synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004642 describes the enzymatic activity of phosphoribosylformylglycinamidine synthase (PFAS), which catalyzes the ATP-dependent conversion of FGAR to FGAM in de novo purine biosynthesis.
• PFAS is a glutamine amidotransferase that couples glutamine hydrolysis to the amidation of FGAR, producing glutamate, ADP, and inorganic phosphate.
• Loss-of-function mutations in PFAS cause a novel inborn error of purine de novo synthesis with neurological and metabolic features.
• PFAS expression is proliferation-linked and is elevated in several cancers, including retinoblastoma, where NSUN2-mediated m5C methylation stabilizes PFAS mRNA.
• Genome-wide CRISPR screening has linked PFAS and nucleotide synthesis to negative regulation of autophagy, highlighting its role in cellular stress responses.
• High-throughput assays and interactome studies provide tools to discover PFAS inhibitors and understand its protein partnerships.
Description
Phosphoribosylformylglycinamidine synthase (PFAS) is a central enzyme in the de novo purine biosynthesis pathway, catalyzing the fourth step that commits FGAR to FGAM formation. This activity, annotated as GO:0004642, is essential for supplying purine nucleotides for DNA and RNA synthesis, making it a key node in cell proliferation and metabolism. Researchers study PFAS because its dysfunction causes a rare inherited metabolic disease and because its overexpression supports tumor growth in several malignancies. Understanding the molecular mechanism, regulation, and disease links of PFAS is therefore critical for both fundamental biology and therapeutic development.
phosphoribosylformylglycinamidine synthase activity At A Glance
| GO ID | GO:0004642 |
|---|---|
| GO term | phosphoribosylformylglycinamidine synthase activity |
| Ontology | molecular_function |
| Synonym | FGAM synthase activity; FGAR amidotransferase activity; FGARAT activity; phosphoribosylformylglycinamidine synthetase activity |
| Major function | Catalyzes the ATP-dependent amidation of FGAR to FGAM using glutamine as the nitrogen donor in de novo purine biosynthesis |
| EC number | 6.3.5.3 |
| Reaction direction | Forward: FGAR + L-glutamine + ATP + H2O -> FGAM + L-glutamate + ADP + 2 H+ + phosphate |
| Cofactors | ATP, Mg2+ (implied by ATP-dependent ligase activity) |
| Pathway | De novo purine biosynthesis (IMP biosynthesis) |
What Is GO:0004642?
GO:0004642 phosphoribosylformylglycinamidine synthase activity is defined as the catalysis of the reaction: N(2)-formyl-N(1)-(5-phospho-D-ribosyl)glycinamide (FGAR) + L-glutamine + ATP + H2O = 2-(formamido)-N(1)-(5-phospho-D-ribosyl)acetamidine (FGAM) + L-glutamate + ADP + 2 H+ + phosphate. In simpler terms, PFAS uses the energy of ATP to transfer an amide group from glutamine to FGAR, forming FGAM, a crucial intermediate in purine biosynthesis.
Why Is phosphoribosylformylglycinamidine synthase activity Important in Cell Biology?
PFAS activity is indispensable for de novo purine synthesis, which provides the building blocks for DNA and RNA. Its proliferation-linked increase makes it a marker of cell growth and a potential target in cancer and immune disorders. Moreover, PFAS deficiency causes a severe metabolic disorder, underscoring its importance in human health.
• PFAS catalyzes a key step in de novo purine biosynthesis, essential for nucleotide supply.
• Its activity increases with cell proliferation, linking it to growth control.
• PFAS mutations cause a novel inborn error of purine metabolism with neurological symptoms.
• PFAS is overexpressed in retinoblastoma and stabilized by NSUN2-mediated m5C methylation.
• Genome-wide CRISPR screens identify PFAS as a negative regulator of autophagy.
• PFAS interacts with multiple proteins, suggesting broader cellular roles.
• High-throughput assays enable discovery of PFAS inhibitors for cancer therapy.
• Bacterial PFAS (e.g., yexA in Bacillus subtilis) is a model for enzyme mechanism studies.
• PFAS is a potential biomarker for proliferation in cancer diagnostics.
• Targeting PFAS may overcome chemoresistance in purine-dependent tumors.
Molecular Mechanism of phosphoribosylformylglycinamidine synthase activity
Substrate Binding and ATP Utilization
In simple terms: PFAS grabs FGAR and ATP, using ATP energy to activate FGAR for amidation.
PFAS binds FGAR and ATP, forming a reactive intermediate that facilitates the transfer of an amide group from glutamine. ATP is hydrolyzed to ADP and phosphate, driving the reaction forward.
Glutamine Hydrolysis and Ammonia Transfer
In simple terms: PFAS breaks down glutamine to release ammonia, which is then added to FGAR.
The enzyme catalyzes the hydrolysis of glutamine to glutamate and ammonia. The ammonia is channeled to the FGAR intermediate, forming FGAM. This glutamine amidotransferase activity is characteristic of PFAS.
Product Formation and Release
In simple terms: PFAS releases FGAM, glutamate, ADP, and phosphate, completing the reaction.
After amidation, PFAS releases FGAM, L-glutamate, ADP, and inorganic phosphate. FGAM then proceeds to the next step in purine biosynthesis.
Structural Organization and Interactome
In simple terms: PFAS is a large enzyme that may work with partner proteins.
PFAS is a multidomain enzyme with separate glutaminase and synthetase domains. Proteomic studies have identified interacting proteins, suggesting it may function in a complex.
Regulation by Proliferation and RNA Modification
In simple terms: PFAS levels go up when cells grow, and its mRNA can be chemically modified to last longer.
PFAS activity increases with cell proliferation. In retinoblastoma, NSUN2-mediated m5C methylation of PFAS mRNA enhances its stability and expression, linking epitranscriptomic regulation to purine synthesis.
Key Genes Involved in GO:0004642 phosphoribosylformylglycinamidine synthase activity
The following genes and proteins are directly or indirectly associated with PFAS activity and purine biosynthesis.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PFAS | Encodes phosphoribosylformylglycinamidine synthase, catalyzing FGAR to FGAM | Mutations cause inborn error; overexpressed in cancers |
| NSUN2 | RNA m5C methyltransferase that stabilizes PFAS mRNA | Regulates PFAS expression in retinoblastoma |
| ATIC | Bifunctional enzyme in purine biosynthesis (IMP cyclohydrolase/AICAR transformylase) | Part of same pathway, potential combination targets |
| GART | Trifunctional enzyme in purine biosynthesis (GARS/AIRS/GART) | Upstream of PFAS, pathway coordination |
| PAICS | Multifunctional enzyme in purine biosynthesis (AIR carboxylase/SAICAR synthetase) | Downstream of PFAS, pathway coordination |
| ADSL | Adenylosuccinate lyase, purine biosynthesis | Defects cause metabolic disorders |
| PPAT | Phosphoribosyl pyrophosphate amidotransferase, first step of purine synthesis | Regulates pathway flux |
| MTHFD1 | Methylenetetrahydrofolate dehydrogenase, folate metabolism | Provides one-carbon units for purine synthesis |
| GLS | Glutaminase, provides glutamine for PFAS | Glutamine metabolism linked to PFAS activity |
| MYC | Oncogene that drives proliferation and nucleotide synthesis | Indirectly upregulates PFAS |
| mTOR | Kinase that promotes anabolic metabolism | May regulate PFAS expression via growth signals |
| ATF4 | Stress-responsive transcription factor | May induce PFAS under amino acid stress |
| yexA | Bacillus subtilis gene required for PFAS activity | Bacterial model for enzyme function |
| PW1 | Imprinted gene involved in cardiac fibrosis | Potential link to purine metabolism? |
| RB1 | Retinoblastoma tumor suppressor | Loss leads to PFAS upregulation via NSUN2 |
| E2F1 | Transcription factor controlling cell cycle | May regulate PFAS promoter |
| CTPS1 | CTP synthase, pyrimidine synthesis | Balances nucleotide pools with PFAS |
| RRM2 | Ribonucleotide reductase, dNTP synthesis | Coordinates with purine synthesis |
How Is phosphoribosylformylglycinamidine synthase activity Regulated?
PFAS activity is regulated at multiple levels. Its expression is proliferation-linked, increasing in dividing cells. In retinoblastoma, NSUN2-mediated m5C methylation of PFAS mRNA enhances its stability, leading to elevated PFAS levels. Additionally, genome-wide CRISPR screening suggests that nucleotide synthesis, including PFAS, negatively regulates autophagy, implying a feedback mechanism. The enzyme may also be subject to allosteric regulation by purine nucleotides, though direct evidence is limited.
phosphoribosylformylglycinamidine synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| PFAS | PFAS deficiency (inborn error of purine synthesis) | Patient-derived fibroblasts, CRISPR KO iPSCs |
| PFAS | Retinoblastoma | RB1-/- retinoblastoma cell lines, xenografts |
| PFAS | Cancer proliferation | Cancer cell lines with PFAS overexpression or KO |
| PFAS | Autophagy regulation | CRISPR KO HeLa cells, autophagy flux assays |
| NSUN2 | Retinoblastoma progression | NSUN2 KO retinoblastoma cells |
PFAS Deficiency: A Novel Inborn Error of Purine Metabolism
Biallelic mutations in PFAS cause a rare metabolic disorder characterized by developmental delay, seizures, and metabolic abnormalities. Zikanova et al. (2025) described clinical, genetic, and metabolic features of PFAS deficiency, highlighting the critical role of this enzyme in human neurodevelopment.
PFAS in Cancer: Retinoblastoma and Beyond
PFAS is overexpressed in retinoblastoma, where NSUN2-mediated m5C methylation stabilizes its mRNA. Knockdown of PFAS suppresses tumor growth, suggesting it as a therapeutic target. Its proliferation-linked activity also makes it a potential target in other cancers.
PFAS and Autophagy Regulation
Genome-wide CRISPR screening revealed that loss of PFAS and other nucleotide synthesis enzymes activates autophagy. This links PFAS to cellular stress responses and suggests that inhibiting PFAS may modulate autophagy in cancer therapy.
From phosphoribosylformylglycinamidine synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does PFAS loss affect purine levels and cell growth? | PFAS knockout cell lines (e.g., HAP1, HeLa) |
| Can a point mutation in PFAS cause enzyme deficiency? | Patient-derived point mutations knocked into cell lines |
| Does PFAS overexpression drive proliferation? | PFAS overexpression in cancer cell lines |
| How does PFAS interact with other proteins? | Endogenous PFAS tagging (knock-in) followed by immunoprecipitation |
| What is the role of PFAS in autophagy? | PFAS knockout with autophagy reporters |
| Can PFAS be targeted by small molecules? | High-throughput enzyme assay with recombinant PFAS |
How to Study the phosphoribosylformylglycinamidine synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay (ADP-Glo) | PFAS activity via ADP production | High-throughput inhibitor screening |
| CRISPR knockout screen | Gene essentiality and pathway interactions | Identifying PFAS role in autophagy |
| LC-MS/MS proteomics | Protein-protein interactions | Mapping PFAS interactome |
| Metabolomics | Purine intermediate levels | Diagnosing PFAS deficiency |
| qRT-PCR | PFAS mRNA expression | Measuring proliferation-linked expression |
| Western blot | PFAS protein levels | Validating knockdown or overexpression |
| Immunofluorescence | Subcellular localization | Determining PFAS localization |
| RNA immunoprecipitation | m5C modification of PFAS mRNA | Studying NSUN2 regulation |
Enzymatic Assays for PFAS Activity
High-throughput assays measure PFAS activity by detecting ADP or phosphate production. Sharma et al. (2025) developed a sensitive assay suitable for inhibitor screening.
CRISPR Screening to Identify PFAS Functions
Genome-wide CRISPR knockout screens can reveal pathways regulated by PFAS. Mimura et al. (2021) identified nucleotide synthesis, including PFAS, as a negative regulator of autophagy using this approach.
Proteomic Interactome Analysis
Affinity purification coupled with mass spectrometry (AP-MS) can identify PFAS-interacting proteins. Lu et al. (2019) mapped the PFAS interactome using LC-MS/MS, uncovering potential new roles.
Metabolic Profiling and Clinical Diagnostics
Targeted metabolomics can quantify purine intermediates in patient samples. Zikanova et al. (2025) used metabolic profiling to characterize PFAS deficiency.
How CRISPR Can Be Used to Study GO:0004642 phosphoribosylformylglycinamidine synthase activity
Knockout
CRISPR knockout of PFAS in cell lines (e.g., HeLa, HAP1) abolishes enzyme activity, reduces purine levels, and can induce autophagy. These models are used to study PFAS essentiality and metabolic rewiring.
Point Mutation
Knocking in patient-derived point mutations (e.g., missense mutations in PFAS) allows functional characterization of enzyme variants and validation of pathogenicity.
Knock-in
Tagged knock-in of PFAS (e.g., GFP or HA tag) enables live-cell imaging, immunoprecipitation, and proteomic studies to understand its localization and interactions.
Overexpression
Overexpressing PFAS in cancer cell lines can model its role in tumor proliferation and test whether increased purine synthesis drives growth or chemoresistance.
How EDITGENE Supports phosphoribosylformylglycinamidine synthase activity Research
Researchers studying phosphoribosylformylglycinamidine synthase activity-related genes often need to determine whether a candidate gene is causally involved in purine metabolism, cancer proliferation, or metabolic disease. EDITGENE provides comprehensive CRISPR services to create precise cellular models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for phosphoribosylformylglycinamidine synthase activity research.
Frequently Asked Questions About phosphoribosylformylglycinamidine synthase activity
What is phosphoribosylformylglycinamidine synthase activity?
It is the enzymatic activity (GO:0004642) of PFAS, which converts FGAR to FGAM in purine biosynthesis using ATP and glutamine.
What genes are involved in phosphoribosylformylglycinamidine synthase activity?
The primary gene is PFAS, but related genes include NSUN2 (regulates PFAS mRNA), ATIC, GART, and PAICS in the purine pathway.
What diseases are associated with PFAS mutations?
PFAS deficiency causes a rare inborn error of purine metabolism with neurological symptoms; PFAS overexpression is linked to retinoblastoma and other cancers.
How is PFAS activity measured?
High-throughput enzymatic assays detect ADP or phosphate production; mass spectrometry can quantify purine intermediates.
Is PFAS a good cancer drug target?
PFAS is proliferation-linked and overexpressed in some cancers, making it a potential target, but further studies are needed.
What is the role of PFAS in autophagy?
Genome-wide CRISPR screens showed that loss of PFAS and nucleotide synthesis activates autophagy, suggesting a regulatory link.
Can CRISPR be used to study PFAS function?
Yes, CRISPR knockout, knock-in, and point mutation models are powerful tools to dissect PFAS roles in cells.
What is the EC number for PFAS?
The EC number is 6.3.5.3.
How is PFAS regulated?
PFAS expression is proliferation-linked and can be stabilized by NSUN2-mediated m5C methylation.
What model organisms are used to study PFAS?
Bacillus subtilis yexA is a bacterial model; human cell lines and patient fibroblasts are used for disease studies.
Conclusion
Phosphoribosylformylglycinamidine synthase activity (GO:0004642) is a critical enzymatic step in de novo purine biosynthesis, with essential roles in cell proliferation, metabolism, and disease. Its dysfunction causes a rare metabolic disorder, while its overexpression contributes to cancer. Continued research using CRISPR models and high-throughput assays will further illuminate PFAS biology and its therapeutic potential.
References
- 2. Sharma N et al.. 2025. A high throughput assay for phosphoribosylformylglycinamidine synthase.. SLAS Discov 31:100212 PMID: 39824442
- 3. Zuo S et al.. 2023. NSUN2-mediated m(5) C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression.. Clin Transl Med 13(5):e1273 PMID: 37228185
- 4. Zikanova M et al.. 2025. Phosphoribosylformylglycinamidine Synthase (PFAS) Deficiency: Clinical, Genetic and Metabolic Characterisation of a Novel Defect in Purine de Novo Synthesis.. J Inherit Metab Dis 48(3):e70041 PMID: 40421664
- 5. Elliott WL et al.. 1984. Proliferation-linked increase in phosphoribosylformylglycinamidine synthetase activity (EC 6.3.5.3).. Cancer Res 44(6):2430-4 PMID: 6722784
- 6. Mimura K et al.. 2021. Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.. J Biol Chem 296:100780 PMID: 34000301
- 7. Lu A et al.. 2019. Protein interactome of the deamidase phosphoribosylformylglycinamidine synthetase (PFAS) by LC-MS/MS.. Biochem Biophys Res Commun 513(3):746-752 PMID: 30987822
- 8. Saxild HH et al.. 2000. The yexA gene product is required for phosphoribosylformylglycinamidine synthetase activity in Bacillus subtilis.. Microbiology (Reading) 146 ( Pt 4):807-814 PMID: 10784038
