GO:0017113 dihydropyrimidine dehydrogenase (NADP+) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0017113 describes the NADP+-dependent catalytic activity of dihydropyrimidine dehydrogenase (DPD), which reduces uracil to 5,6-dihydrouracil and thymine to 5,6-dihydrothymine.
• DPD is the rate-limiting enzyme of the pyrimidine degradation pathway and is encoded by the DPYD gene in humans.
• DPD activity is clinically critical because it catabolizes approximately 80% of administered 5-fluorouracil (5-FU), and low activity causes severe fluoropyrimidine toxicity.
• DPYD genetic variants, such as DPYD*2A, reduce DPD activity and are used to predict 5-FU toxicity risk.
• Endogenous dihydropyrimidine metabolites can serve as biomarkers for DPD activity, enabling non-invasive assessment.
• CRISPR-based knockout, point-mutation, and knock-in models are essential for dissecting DPYD variant function and DPD-related drug responses.
Description
Dihydropyrimidine dehydrogenase (NADP+) activity (GO:0017113) is a molecular function defined as the catalysis of the reaction 5,6-dihydrouracil + NADP+ = uracil + NADPH + H+. This activity is carried out by the enzyme dihydropyrimidine dehydrogenase (DPD), which also catalyzes the analogous reduction of thymine to 5,6-dihydrothymine. DPD is the first and rate-limiting enzyme in the pyrimidine degradation pathway, and its activity determines the balance between pyrimidine salvage and catabolism. Because pyrimidines are essential for nucleic acid synthesis and cellular metabolism, DPD dysfunction has broad physiological consequences.
dihydropyrimidine dehydrogenase (NADP+) activity At A Glance
| GO ID | GO:0017113 |
|---|---|
| GO term | dihydropyrimidine dehydrogenase (NADP+) activity |
| Ontology | molecular_function |
| Synonym | dihydropyrimidine dehydrogenase activity; DPD; DHPDH; dihydrouracil dehydrogenase (NADP+) |
| Major function | Catalyzes the NADP+-dependent reduction of uracil to 5,6-dihydrouracil and thymine to 5,6-dihydrothymine |
| Reaction | 5,6-dihydrouracil + NADP+ = uracil + NADPH + H+ |
| Cofactor | NADP+/NADPH |
| Pathway | Pyrimidine degradation (catabolism) |
| Subcellular location | Cytosol |
What Is GO:0017113?
GO:0017113 describes the catalytic activity of an enzyme that transfers electrons from NADPH to uracil (or thymine), reducing the 5,6 double bond to produce 5,6-dihydrouracil (or 5,6-dihydrothymine) and NADP+. The reaction is reversible in vitro but physiologically favors pyrimidine catabolism. This activity is synonymous with dihydropyrimidine dehydrogenase activity, DHPDH, and DPD.
Why Is dihydropyrimidine dehydrogenase (NADP+) activity Important in Cell Biology?
DPD activity is a critical determinant of fluoropyrimidine chemotherapy outcomes. Approximately 80% of administered 5-fluorouracil (5-FU) is catabolized by DPD, and patients with low DPD activity are at high risk for severe, sometimes fatal, toxicity. Measuring DPD activity and identifying DPYD variants are therefore central to personalized dosing of 5-FU and capecitabine. Beyond oncology, DPD deficiency causes pyrimidinemia and neurological symptoms, highlighting its role in normal pyrimidine homeostasis.
• DPD is the rate-limiting enzyme of pyrimidine catabolism, controlling uracil and thymine levels.
• DPD activity predicts 5-FU toxicity and response in cancer patients.
• DPYD variants, such as DPYD*2A, reduce DPD activity and increase fluoropyrimidine toxicity risk.
• Endogenous dihydropyrimidine metabolites can serve as biomarkers for DPD activity.
• DPD deficiency is associated with neurological disorders and pyrimidinemia.
• DPD is a target for modulating 5-FU efficacy in colorectal and other cancers.
• Assessing DPD activity helps guide dose adjustments for capecitabine and 5-FU.
• DPD expression varies among tissues and individuals, affecting drug metabolism.
What Happens During dihydropyrimidine dehydrogenase (NADP+) activity?
Substrate binding and reduction
In simple terms: DPD grabs uracil or thymine and uses NADPH to break a double bond, turning them into dihydropyrimidines.
DPD binds uracil or thymine in its active site and catalyzes the NADPH-dependent reduction of the 5,6 double bond, yielding 5,6-dihydrouracil or 5,6-dihydrothymine, respectively. This reaction is the first committed step in pyrimidine catabolism.
NADPH oxidation and electron transfer
In simple terms: NADPH gives up electrons to the substrate, becoming NADP+.
The enzyme uses NADPH as an electron donor, oxidizing it to NADP+ while reducing the pyrimidine ring. This redox reaction is essential for the subsequent steps of pyrimidine degradation.
Role in pyrimidine homeostasis
In simple terms: By breaking down uracil and thymine, DPD keeps pyrimidine levels balanced.
DPD activity regulates the pool of free pyrimidines, preventing accumulation of uracil and thymine, which can be toxic at high levels. This homeostatic function is critical for normal cellular metabolism.
Impact on 5-fluorouracil metabolism
In simple terms: DPD also destroys 5-FU, so low DPD means more drug stays active and can cause harm.
DPD catabolizes approximately 80% of administered 5-FU to inactive dihydrofluorouracil, thereby limiting its antitumor activity and toxicity. Consequently, DPD activity inversely correlates with 5-FU bioavailability and toxicity risk.
Key Genes Involved in GO:0017113 dihydropyrimidine dehydrogenase (NADP+) activity
The following genes and proteins are directly involved in dihydropyrimidine dehydrogenase (NADP+) activity and its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DPYD | Encodes dihydropyrimidine dehydrogenase, the enzyme catalyzing GO:0017113 | Mutations cause DPD deficiency and 5-FU toxicity |
| DPYS | Encodes dihydropyrimidinase, the second enzyme in pyrimidine degradation | Downstream of DPD; mutations cause dihydropyrimidinuria |
| UPB1 | Encodes beta-ureidopropionase, the third enzyme in pyrimidine degradation | Defects lead to neurological symptoms |
| TYMP | Encodes thymidine phosphorylase, involved in thymidine catabolism | Affects pyrimidine pool and 5-FU activation |
| UCK2 | Encodes uridine-cytidine kinase 2, a pyrimidine salvage enzyme | Balances salvage versus catabolism |
| CAD | Encodes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase | De novo pyrimidine synthesis, opposite to DPD catabolism |
| UMPS | Encodes uridine monophosphate synthetase | Pyrimidine synthesis; interacts with DPD pathway |
| CMPK1 | Encodes cytidine monophosphate kinase | Pyrimidine nucleotide metabolism |
| NT5C | Encodes 5',3'-nucleotidase, cytosolic | Pyrimidine salvage |
| SLC29A1 | Encodes equilibrative nucleoside transporter 1 | Uptake of pyrimidine nucleosides |
| SLC28A1 | Encodes concentrative nucleoside transporter 1 | Uptake of pyrimidine nucleosides |
| TP53 | Tumor suppressor, frequently mutated in cancers | May influence 5-FU response |
| MTHFR | Methylenetetrahydrofolate reductase | Folate metabolism, modifies 5-FU efficacy |
| TYMS | Thymidylate synthase, target of 5-FU | Expression affects 5-FU response |
| GSTP1 | Glutathione S-transferase P1 | Drug detoxification, may affect 5-FU toxicity |
| ABCB1 | ATP-binding cassette subfamily B member 1 | Drug efflux, influences 5-FU resistance |
How Is dihydropyrimidine dehydrogenase (NADP+) activity Regulated?
DPD activity is regulated at multiple levels. DPYD gene expression is influenced by genetic variants, including the splice-site mutation DPYD*2A, which reduces enzyme activity. Transcriptional regulation by transcription factors such as Sp1 and NF-κB has been reported, but the exact mechanisms remain incompletely understood. Additionally, DPD activity can be modulated by post-translational modifications and by the availability of NADPH. In cancer cells, DPD expression is often upregulated, contributing to 5-FU resistance.
dihydropyrimidine dehydrogenase (NADP+) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DPYD | DPD deficiency, 5-FU toxicity | DPYD knockout cell line (e.g., HCT116) |
| DPYD | DPYD*2A splice variant | Point-mutation knock-in of DPYD*2A in iPSCs |
| DPYS | Dihydropyrimidinuria | DPYS knockout HEK293 cells |
| UPB1 | Beta-ureidopropionase deficiency | UPB1 knockout HepG2 cells |
| TYMS | 5-FU resistance | TYMS overexpression in colorectal cancer cells |
DPD deficiency and fluoropyrimidine toxicity
Inherited DPD deficiency, caused by biallelic DPYD mutations, leads to reduced or absent DPD activity and severe toxicity upon 5-FU administration. Patients may experience myelosuppression, mucositis, and neurotoxicity, sometimes fatal. Preemptive genotyping of DPYD variants is recommended to adjust 5-FU doses.
DPD in cancer and 5-FU resistance
High DPD expression in tumors is associated with resistance to 5-FU, as the drug is rapidly catabolized. Conversely, low DPD activity in tumors may enhance 5-FU efficacy but increase systemic toxicity. DPD inhibitors are being explored to overcome resistance.
Neurological manifestations of DPD deficiency
DPD deficiency can cause pyrimidinemia and neurological symptoms such as seizures, developmental delay, and microcephaly, due to accumulation of uracil and thymine. These symptoms can occur even without 5-FU exposure.
From dihydropyrimidine dehydrogenase (NADP+) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does DPYD loss alter 5-FU sensitivity? | DPYD knockout in HCT116 or SW480 cells |
| How does DPYD*2A affect DPD activity? | Point-mutation knock-in of DPYD*2A in HEK293T cells |
| Can DPD activity be restored by wild-type DPYD? | Knock-in of wild-type DPYD in DPYD-null cells |
| What is the effect of DPD overexpression on 5-FU resistance? | DPYD overexpression in colorectal cancer cells |
| Can endogenous metabolites predict DPD activity? | Metabolomic profiling of DPYD knockout cells |
| Does DPD deficiency cause neurological phenotypes? | DPYD knockout in iPSC-derived neurons |
How to Study the dihydropyrimidine dehydrogenase (NADP+) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC-based enzyme assay | DPD catalytic activity | Assessing DPYD variant function |
| LC-MS/MS metabolomics | Uracil and dihydrouracil levels | Predicting DPD activity in patients |
| Sanger sequencing | DPYD mutations | Genotyping DPYD*2A and other variants |
| CRISPR-Cas9 knockout | Gene function loss | Creating DPYD-null cell models |
| CRISPR knock-in | Specific mutation introduction | Modeling DPYD*2A |
| Western blot | DPD protein expression | Validating knockout or overexpression |
| RNA-seq | Transcriptome changes | Identifying DPD-regulated pathways |
| Cell viability assay | 5-FU sensitivity | Linking DPD activity to drug response |
Enzyme activity assays
DPD activity is typically measured using radiolabeled uracil or thymine and HPLC to quantify 5,6-dihydrouracil formation. These assays are used to assess the impact of DPYD variants on catalytic function.
Genotyping and variant analysis
DPYD variants are identified by PCR and sequencing, including targeted assays for DPYD*2A, DPYD*13, and other clinically relevant alleles. Genotyping helps predict DPD activity and 5-FU toxicity risk.
Metabolomics for DPD activity
Endogenous dihydropyrimidine metabolites, such as uracil and dihydrouracil, can be quantified by LC-MS/MS to estimate DPD activity in patient plasma. This approach offers a non-invasive biomarker for DPD status.
CRISPR-based functional studies
CRISPR-Cas9 knockout, point-mutation knock-in, and overexpression models are used to dissect the functional consequences of DPYD variants and to study DPD-related drug responses.
How CRISPR Can Be Used to Study GO:0017113 dihydropyrimidine dehydrogenase (NADP+) activity
Knockout
CRISPR-Cas9 knockout of DPYD in cancer cell lines (e.g., HCT116) abolishes DPD activity, leading to increased sensitivity to 5-FU and accumulation of uracil. These models are valuable for studying DPD deficiency and drug toxicity.
Point Mutation
Point mutations such as DPYD*2A (splice-site) or DPYD*13 (missense) can be introduced using CRISPR base editing or homology-directed repair to model clinical variants. These models help determine the functional impact of specific alleles on DPD activity.
Knock-in
Knock-in of wild-type or mutant DPYD into a safe locus or the endogenous locus allows controlled expression and functional rescue experiments. This is useful for validating variant pathogenicity.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of DPYD can increase DPD activity, modeling 5-FU resistance in cancer cells. Such models help test DPD inhibitors as chemosensitizers.
How EDITGENE Supports dihydropyrimidine dehydrogenase (NADP+) activity Research
Researchers studying dihydropyrimidine dehydrogenase (NADP+) activity-related genes often need to determine whether a candidate gene is causally involved in DPD function, drug metabolism, or disease. EDITGENE provides custom CRISPR cell models and screening services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for dihydropyrimidine dehydrogenase (NADP+) activity research.
Frequently Asked Questions About dihydropyrimidine dehydrogenase (NADP+) activity
What is dihydropyrimidine dehydrogenase (NADP+) activity?
It is the enzyme activity that catalyzes the NADP+-dependent reduction of uracil to 5,6-dihydrouracil and thymine to 5,6-dihydrothymine, encoded by GO:0017113.
What gene encodes dihydropyrimidine dehydrogenase?
The DPYD gene encodes the human enzyme dihydropyrimidine dehydrogenase.
What is the role of DPD in 5-fluorouracil metabolism?
DPD catabolizes approximately 80% of administered 5-FU to inactive metabolites, thereby limiting its efficacy and toxicity.
How is DPD activity measured?
DPD activity is measured using HPLC-based enzyme assays with radiolabeled uracil or by LC-MS/MS quantification of endogenous dihydropyrimidine metabolites.
What are the clinical implications of DPD deficiency?
DPD deficiency causes severe, potentially fatal toxicity to 5-FU and capecitabine, and can also cause neurological symptoms.
Which DPYD variants affect DPD activity?
Common variants include DPYD*2A (splice-site), DPYD*13 (missense), and DPYD*9A, which reduce enzyme activity.
Can endogenous metabolites predict DPD activity?
Yes, plasma uracil and dihydrouracil levels correlate with DPD activity and can serve as biomarkers.
What diseases are associated with DPD deficiency?
DPD deficiency is linked to fluoropyrimidine toxicity, pyrimidinemia, and neurological disorders.
How can CRISPR help study DPD function?
CRISPR knockout, point mutation, and knock-in models allow functional analysis of DPYD variants and their impact on drug response.
What is the GO term for dihydropyrimidine dehydrogenase activity?
The Gene Ontology term is GO:0017113, dihydropyrimidine dehydrogenase (NADP+) activity.
Conclusion
Dihydropyrimidine dehydrogenase (NADP+) activity (GO:0017113) is a central molecular function in pyrimidine catabolism and a key determinant of fluoropyrimidine drug response. Understanding its genetic regulation and functional consequences is essential for personalized cancer therapy and for diagnosing DPD deficiency. CRISPR-based models and advanced metabolomics provide powerful tools to dissect DPD biology and improve patient outcomes.
References
- 1. Offer SM et al.. 2014. Comparative functional analysis of DPYD variants of potential clinical relevance to dihydropyrimidine dehydrogenase activity.. Cancer Res 74(9):2545-54 PMID: 24648345
- 2. Forouzesh DC et al.. 2021. Mammalian dihydropyrimidine dehydrogenase.. Arch Biochem Biophys 714:109066 PMID: 34717904
- 3. Kang J et al.. 2022. Endogenous metabolic markers for predicting the activity of dihydropyrimidine dehydrogenase.. Clin Transl Sci 15(5):1104-1111 PMID: 34863048
- 4. Ogura K. 2006. [Dihydropyrimidine dehydrogenase activity and its genetic aberrations].. Gan To Kagaku Ryoho 33(8):1041-8 PMID: 16912518
- 5. Collie-Duguid ES et al.. 2000. Estimation of dihydropyrimidine dehydrogenase activity: does it have a role in cancer therapy?. Ann Oncol 11(3):255-7 PMID: 10811489
- 6. Kubota T. 2003. 5-fluorouracil and dihydropyrimidine dehydrogenase.. Int J Clin Oncol 8(3):127-31 PMID: 12851835
- 7. Gardiner SJ et al.. 2002. The effect of dihydropyrimidine dehydrogenase deficiency on outcomes with fluorouracil.. Adverse Drug React Toxicol Rev 21(1-2):1-16 PMID: 12140902
- 8. Diasio RB et al.. 1994. Dihydropyrimidine dehydrogenase activity and fluorouracil chemotherapy.. J Clin Oncol 12(11):2239-42 PMID: 7964937
