GO:0004643 phosphoribosylaminoimidazolecarboxamide formyltransferase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004643 describes the enzymatic activity that transfers a formyl group from 10-formyltetrahydrofolate to 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR), producing 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide and tetrahydrofolate.
• This activity is also known as AICAR transformylase and is a folate-dependent step in de novo purine biosynthesis.
• The reaction is a validated target of antifolate drugs such as methotrexate and its polyglutamated metabolites, which inhibit the enzyme.
• Genetic variation in folate and purine pathway genes, including those related to this activity, has been studied as predictors of methotrexate response in rheumatoid arthritis.
• Non-steroidal anti-inflammatory drugs can also inhibit folate-dependent enzymes, suggesting broader pharmacological interactions.
• Loss of the related atic gene in zebrafish impairs muscle homeostasis and mitochondrial function, linking purine biosynthesis to tissue maintenance.
Description
GO:0004643, phosphoribosylaminoimidazolecarboxamide formyltransferase activity, is a molecular function that catalyzes a folate-dependent formylation step in de novo purine biosynthesis. The reaction converts 10-formyltetrahydrofolate and 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR) into tetrahydrofolate and 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide. This activity is commonly referred to as AICAR transformylase and is essential for the production of purine nucleotides that support DNA, RNA, and ATP synthesis. Because it consumes a folate cofactor, the enzyme is mechanistically linked to one-carbon metabolism and is a direct target of antifolate drugs such as methotrexate. Researchers study this activity to understand how cells balance purine supply with folate availability, and how pharmacological or genetic perturbation of the pathway affects proliferation, immune function, and tissue repair. Inhibition of AICAR transformylase by methotrexate polyglutamates provided early evidence that the drug's anti-inflammatory and antiproliferative effects extend beyond dihydrofolate reductase inhibition. In parallel, clinical pharmacogenetic studies have examined variants in folate and purine pathway genes as predictors of methotrexate response in rheumatoid arthritis, underscoring the translational relevance of this enzymatic step. This article summarizes the definition, mechanism, key genes, disease links, and experimental models for GO:0004643, with a focus on how CRISPR-based cell models and functional genomics can be used to dissect its role in health and disease.
phosphoribosylaminoimidazolecarboxamide formyltransferase activity At A Glance
| GO ID | GO:0004643 |
|---|---|
| GO term | phosphoribosylaminoimidazolecarboxamide formyltransferase activity |
| Ontology | molecular_function |
| Synonym | AICAR transformylase activity; 10-formyltetrahydrofolate:5'-phosphoribosyl-5-amino-4-imidazolecarboxamide formyltransferase activity; 5-amino-4-imidazolecarboxamide ribonucleotide transformylase activity |
| Major function | Catalyzes a folate-dependent formylation step in de novo purine biosynthesis |
| Reaction | 10-formyltetrahydrofolate + 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide = tetrahydrofolate + 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide |
| Substrates | 10-formyltetrahydrofolate and 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR) |
| Products | Tetrahydrofolate and 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide |
| Pathway context | De novo purine biosynthesis; folate one-carbon metabolism |
| Pharmacological relevance | Inhibited by methotrexate and dihydrofolic acid polyglutamates |
What Is GO:0004643?
In simple terms, GO:0004643 is the activity of an enzyme that attaches a formyl group to a purine precursor using a folate carrier. The official definition states: Catalysis of the reaction: 10-formyltetrahydrofolate + 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide = tetrahydrofolate + 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide. This reaction is a mid-pathway step in de novo purine biosynthesis, and the enzyme is often called AICAR transformylase. The activity depends on 10-formyltetrahydrofolate as the formyl donor and releases tetrahydrofolate, thereby coupling purine synthesis to one-carbon folate metabolism.
Why Is phosphoribosylaminoimidazolecarboxamide formyltransferase activity Important in Cell Biology?
GO:0004643 is important because it connects folate metabolism to purine nucleotide production, two processes that are essential for cell proliferation and are frequently targeted in cancer and autoimmune disease therapy. The enzyme is directly inhibited by methotrexate polyglutamates, which helps explain the drug's mechanism of action beyond dihydrofolate reductase. Genetic and clinical studies have repeatedly examined folate and purine pathway variants as predictors of methotrexate response in rheumatoid arthritis, making this activity a focal point for pharmacogenomic research. In addition, disruption of the purine biosynthetic pathway in model organisms can affect muscle and mitochondrial function, indicating broader physiological roles.
• Provides a key formylation step in de novo purine biosynthesis, supporting DNA, RNA, and ATP production.
• Couples purine synthesis to folate one-carbon metabolism through consumption of 10-formyltetrahydrofolate.
• Is a direct target of methotrexate and its polyglutamated metabolites, linking the activity to antifolate drug action.
• Genetic variants in folate and purine pathway genes have been investigated as predictors of methotrexate response in rheumatoid arthritis.
• Non-steroidal anti-inflammatory drugs can inhibit folate-dependent enzymes, suggesting potential drug-drug interactions at this step.
• Loss of the related atic gene in zebrafish impairs skeletal muscle homeostasis and oxidative phosphorylation.
• The activity is relevant to proliferative diseases such as cancer and to immune-mediated inflammatory diseases.
• It provides a mechanistic bridge between one-carbon metabolism and nucleotide biosynthesis for metabolic research.
• It is a candidate target for therapeutic modulation in conditions where purine supply or folate flux is dysregulated.
• It offers a defined biochemical readout for testing CRISPR-engineered cell models of purine pathway genes.
Molecular Mechanism of phosphoribosylaminoimidazolecarboxamide formyltransferase activity
Substrate recognition and binding
In simple terms: The enzyme first grabs its two starting materials: a folate carrier and a purine precursor.
The activity uses 10-formyltetrahydrofolate as the formyl donor and 5'-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR) as the acceptor substrate. The enzyme binds both substrates and positions the formyl group for transfer to the amino group of AICAR. This step is part of the de novo purine biosynthetic pathway and is sensitive to inhibition by antifolates such as methotrexate and dihydrofolic acid polyglutamates.
Formyl transfer and product release
In simple terms: The enzyme moves a chemical tag from the folate carrier onto the purine precursor, then releases the finished product.
Catalysis transfers the formyl group from 10-formyltetrahydrofolate to AICAR, yielding 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide and tetrahydrofolate. The reaction is defined by GO:0004643 and is often referred to as AICAR transformylase activity. Because the reaction consumes a folate cofactor, its rate can be influenced by cellular folate status and by drugs that interfere with folate metabolism.
Cofactor dependence and folate cycle coupling
In simple terms: The enzyme depends on a folate-based carrier and is tied to the broader folate cycle.
The formyl donor 10-formyltetrahydrofolate is a central one-carbon carrier, so this activity is mechanistically coupled to folate metabolism. Inhibition of the enzyme by methotrexate polyglutamates demonstrates that the folate cycle and purine biosynthesis are pharmacologically linked. Non-steroidal anti-inflammatory drugs can also inhibit folate-dependent enzymes, suggesting that this step may be affected by additional pharmacological inputs.
Pathway context and regulation
In simple terms: This reaction sits in the middle of the purine-building assembly line and can be tuned by pathway demand.
The activity is a mid-pathway step in de novo purine biosynthesis, which supplies purine nucleotides for DNA, RNA, and energy metabolism. Genetic and clinical studies have examined variants in folate and purine pathway genes as predictors of methotrexate response, indicating that pathway context and genetic background can influence the effective activity of this step. In zebrafish, loss of the related atic gene affects skeletal muscle and oxidative phosphorylation, highlighting the physiological importance of purine pathway integrity.
Key Genes Involved in GO:0004643 phosphoribosylaminoimidazolecarboxamide formyltransferase activity
The following genes and proteins are directly or functionally associated with GO:0004643 and its pathway context.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ATIC | Encodes a bifunctional enzyme with AICAR transformylase and IMP cyclohydrolase activities | Central to de novo purine biosynthesis; related gene studied in zebrafish muscle atrophy |
| GART | Encodes a trifunctional enzyme in purine biosynthesis | Pathway context for folate-dependent purine synthesis |
| MTHFR | Folate metabolism enzyme | Studied as a predictor of methotrexate response in rheumatoid arthritis |
| MTR | Methionine synthase, folate cycle | Candidate gene in methotrexate pharmacogenetics |
| MTRR | Methionine synthase reductase | Folate pathway gene examined in methotrexate response studies |
| SLC19A1 | Folate transporter | Influences intracellular folate availability for folate-dependent enzymes |
| ABCB1 | Drug transporter | May affect methotrexate pharmacokinetics and response |
| FPGS | Folylpolyglutamate synthetase | Determines formation of methotrexate polyglutamates that inhibit the enzyme |
| GGH | Gamma-glutamyl hydrolase | Affects polyglutamate turnover and antifolate activity |
| DHFR | Dihydrofolate reductase | Folate cycle enzyme and methotrexate target |
| TYMS | Thymidylate synthase | Folate-dependent enzyme in nucleotide synthesis |
| ATIC (zebrafish atic) | Purine biosynthesis enzyme | Knockout causes skeletal muscle atrophy and oxidative phosphorylation changes |
| ADSL | Purine biosynthesis enzyme | Pathway context for de novo purine synthesis |
| PAICS | Purine biosynthesis enzyme | Pathway context for de novo purine synthesis |
| PPAT | Purine biosynthesis enzyme | Pathway context for de novo purine synthesis |
| GART (alternative) | Purine biosynthesis enzyme | Pathway context for de novo purine synthesis |
| MTHFD1 | One-carbon metabolism | Supplies 10-formyltetrahydrofolate for folate-dependent reactions |
| SHMT1 | One-carbon metabolism | Contributes to folate one-carbon pools |
How Is phosphoribosylaminoimidazolecarboxamide formyltransferase activity Regulated?
The activity is regulated at the level of substrate availability, particularly the supply of 10-formyltetrahydrofolate from one-carbon metabolism, and by the presence of antifolate drugs such as methotrexate and its polyglutamated metabolites that inhibit the enzyme. Non-steroidal anti-inflammatory drugs can also inhibit folate-dependent enzymes, suggesting additional pharmacological modulation. Genetic variation in folate and purine pathway genes has been associated with differences in methotrexate response in rheumatoid arthritis, indicating that inherited factors can influence pathway flux. In zebrafish, loss of the related atic gene alters oxidative phosphorylation and muscle homeostasis, showing that pathway integrity is required for tissue maintenance.
phosphoribosylaminoimidazolecarboxamide formyltransferase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ATIC | Purine biosynthesis and muscle homeostasis | Zebrafish atic knockout |
| MTHFR | Methotrexate response in rheumatoid arthritis | Human cell lines with point mutations |
| SLC19A1 | Folate transport and drug response | Knockout cell models |
| FPGS | Methotrexate polyglutamation and enzyme inhibition | Knock-in/overexpression models |
| DHFR | Folate cycle and antifolate therapy | CRISPR knockout and point mutation models |
Rheumatoid arthritis and methotrexate response
Methotrexate is a first-line therapy for rheumatoid arthritis, and its mechanism includes inhibition of folate-dependent enzymes such as AICAR transformylase. Clinical pharmacogenetic studies have evaluated variants in folate and purine pathway genes as predictors of methotrexate nonresponse or response in rheumatoid arthritis patients. These studies highlight the potential for genetic markers related to GO:0004643 pathway genes to inform treatment stratification.
Cancer and antifolate therapy
Because the activity supports de novo purine biosynthesis, it is relevant to proliferating cancer cells that require high nucleotide flux. Methotrexate and dihydrofolic acid polyglutamates inhibit the enzyme, providing a rationale for antifolate therapy in malignancies. Targeting this step may affect purine supply and cell proliferation.
Muscle homeostasis and mitochondrial function
In zebrafish, knockout of the atic gene, which is related to the purine biosynthetic pathway, causes skeletal muscle atrophy and changes in oxidative phosphorylation. This model links purine pathway integrity to muscle maintenance and mitochondrial function, suggesting that related enzymes may influence tissue energetics.
Drug interactions and folate metabolism
Non-steroidal anti-inflammatory drugs can inhibit folate-dependent enzymes, which may affect the activity of enzymes like AICAR transformylase. This raises the possibility of drug-drug interactions in patients receiving antifolates and anti-inflammatory agents.
From phosphoribosylaminoimidazolecarboxamide formyltransferase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of ATIC affect purine nucleotide levels? | ATIC knockout cell line |
| Does a specific ATIC point mutation alter enzyme activity? | Point-mutation knock-in cell line |
| Can tagged ATIC be used to monitor localization? | Tagged knock-in cell line |
| Does overexpression of ATIC increase purine flux? | Overexpression cell line |
| Which genes modify methotrexate response? | CRISPR library screening in cell lines |
| Does atic loss affect muscle mitochondria? | Zebrafish atic knockout |
How to Study the phosphoribosylaminoimidazolecarboxamide formyltransferase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | AICAR transformylase activity | Testing inhibition by antifolates |
| CRISPR knockout | Loss-of-function effects | Testing gene requirement in purine synthesis |
| Point-mutation knock-in | Effect of specific variants | Validating pharmacogenetic alleles |
| Metabolomics | Purine and folate metabolite levels | Assessing pathway flux |
| RNA-seq | Transcriptional changes | Pathway response to perturbation |
| Proteomics | Protein expression and interactions | Identifying pathway components |
| Zebrafish models | Tissue-level phenotypes | Muscle and mitochondrial studies |
| Pharmacogenetic modeling | Genotype-response associations | Predicting methotrexate response |
Enzymatic activity assays
Direct measurement of AICAR transformylase activity can be performed using substrate-based assays that monitor the conversion of 10-formyltetrahydrofolate and AICAR to tetrahydrofolate and the formylated product. Such assays are useful for testing inhibition by methotrexate polyglutamates.
CRISPR knockout and point-mutation models
CRISPR-Cas9 can generate knockout or precise point-mutation cell lines in genes such as ATIC, MTHFR, and SLC19A1 to test their roles in purine biosynthesis and drug response. These models allow causal testing of variants identified in pharmacogenetic studies.
Metabolomics and nucleotide profiling
Mass spectrometry-based metabolomics can quantify purine intermediates and folate species to assess pathway flux. This approach can reveal how genetic or pharmacological perturbations affect the reaction catalyzed by GO:0004643.
Pharmacogenomic association studies
Clinical studies have used candidate-gene and pharmacogenetic models to test whether variants in folate and purine pathway genes predict methotrexate response in rheumatoid arthritis. These methods help link genotype to therapeutic outcome.
How CRISPR Can Be Used to Study GO:0004643 phosphoribosylaminoimidazolecarboxamide formyltransferase activity
Knockout
CRISPR knockout of ATIC or related purine pathway genes can be used to determine whether the enzyme is required for cell proliferation and purine nucleotide production. Such models complement zebrafish atic knockout studies that show muscle atrophy and oxidative phosphorylation changes.
Point Mutation
Point-mutation knock-in can recreate specific variants in folate and purine pathway genes identified in pharmacogenetic studies, allowing functional testing of their impact on methotrexate response.
Knock-in
Tagged knock-in of ATIC can enable localization and interaction studies, helping to define where the enzyme acts within the cell and how it is regulated.
Overexpression
Overexpression of ATIC or other pathway genes can test whether increased enzyme levels alter purine flux or drug sensitivity, providing a gain-of-function counterpart to knockout models.
How EDITGENE Supports phosphoribosylaminoimidazolecarboxamide formyltransferase activity Research
Researchers studying phosphoribosylaminoimidazolecarboxamide formyltransferase activity-related genes often need to determine whether a candidate gene is causally involved in purine biosynthesis, folate metabolism, or drug response. EDITGENE provides CRISPR-based cell models and screening services to enable these functional studies.
Contact EDITGENE today to design your custom CRISPR model for phosphoribosylaminoimidazolecarboxamide formyltransferase activity research.
Frequently Asked Questions About phosphoribosylaminoimidazolecarboxamide formyltransferase activity
What is GO:0004643?
GO:0004643 is the Gene Ontology molecular function term for phosphoribosylaminoimidazolecarboxamide formyltransferase activity, also known as AICAR transformylase, which catalyzes a folate-dependent step in purine biosynthesis.
What does phosphoribosylaminoimidazolecarboxamide formyltransferase activity do?
It transfers a formyl group from 10-formyltetrahydrofolate to AICAR, producing tetrahydrofolate and 5'-phosphoribosyl-5-formamido-4-imidazolecarboxamide.
What genes are involved in phosphoribosylaminoimidazolecarboxamide formyltransferase activity?
ATIC encodes the enzyme with this activity; related pathway genes include GART, MTHFR, MTR, MTRR, SLC19A1, FPGS, GGH, DHFR, TYMS, and MTHFD1.
Is AICAR transformylase the same as GO:0004643?
Yes, AICAR transformylase activity is a synonym for GO:0004643.
How is this enzyme inhibited by methotrexate?
Methotrexate and dihydrofolic acid polyglutamates inhibit phosphoribosylaminoimidazolecarboxamide transformylase, contributing to the drug's effects.
Why is this activity important in rheumatoid arthritis?
It is part of the folate and purine pathway targeted by methotrexate, and genetic variants in related genes have been studied as predictors of treatment response.
Can CRISPR be used to study GO:0004643?
Yes, CRISPR knockout, point-mutation knock-in, and overexpression models can test the function of ATIC and related genes in purine biosynthesis and drug response.
What diseases are linked to this activity?
It is linked to rheumatoid arthritis through methotrexate response and to cancer through antifolate therapy; related pathway loss affects muscle in zebrafish.
What experimental models are used for this pathway?
Enzymatic assays, CRISPR cell models, metabolomics, and zebrafish atic knockout are used to study this pathway.
Where can I get CRISPR cell models for ATIC and related genes?
EDITGENE provides knockout, point-mutation, knock-in, overexpression, and library screening services for purine and folate pathway genes.
Conclusion
GO:0004643, phosphoribosylaminoimidazolecarboxamide formyltransferase activity, is a folate-dependent enzymatic step in de novo purine biosynthesis that is directly inhibited by methotrexate polyglutamates. Its pathway context is relevant to rheumatoid arthritis pharmacogenetics and antifolate cancer therapy, and related genes have been studied in clinical and model organism research. CRISPR-based cell models offer a powerful way to test causal roles of ATIC and related genes in purine metabolism and drug response.
References
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- 3. Singh A et al.. 2021. Polymorphism of genes involved in methotrexate pathway: Predictors of response to methotrexate therapy in Indian rheumatoid arthritis patients.. Int J Rheum Dis 24(5):654-662 PMID: 33780152
- 4. Wessels JA et al.. 2007. A clinical pharmacogenetic model to predict the efficacy of methotrexate monotherapy in recent-onset rheumatoid arthritis.. Arthritis Rheum 56(6):1765-75 PMID: 17530705
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- 6. Baggott JE et al.. 1992. Inhibition of folate-dependent enzymes by non-steroidal anti-inflammatory drugs.. Biochem J 282 ( Pt 1)(Pt 1):197-202 PMID: 1540135
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