GO:0047847 deoxyuridine phosphorylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047847 (deoxyuridine phosphorylase activity) is a molecular function that catalyzes the phosphorolysis of 2'-deoxyuridine to 2-deoxy-alpha-D-ribose 1-phosphate and uracil.
• The enzyme is a pyrimidine nucleoside phosphorylase with specificity for deoxyuridine and related analogs such as 5-fluoro-2'-deoxyuridine.
• Its activity is distinct from uridine phosphorylase and is inhibited by 5-azauracil in cell-free extracts.
• Deoxyuridine phosphorylase activity is important for pyrimidine salvage and for modulating the cytotoxicity of fluoropyrimidine drugs [2,8].
• Altered deoxyuridine phosphorylase activity has been observed in regenerating rat liver and in Mycoplasma mycoides, indicating roles in proliferation and microbial metabolism [5,6].
• CRISPR-based knockout, point-mutation, knock-in, and overexpression models enable precise interrogation of deoxyuridine phosphorylase function in health and disease.
Description
Deoxyuridine phosphorylase activity (GO:0047847) is a molecular function that catalyzes the reversible phosphorolysis of 2'-deoxyuridine into 2-deoxy-alpha-D-ribose 1-phosphate and uracil. This reaction is a key step in the salvage pathway of pyrimidine deoxyribonucleosides, allowing cells to recycle deoxyuridine for nucleotide biosynthesis or to degrade it when excess. The enzyme belongs to the family of pyrimidine nucleoside phosphorylases and exhibits specificity for deoxyuridine over uridine in some organisms and tissues [3,5]. Because deoxyuridine phosphorylase can also cleave 5-fluoro-2'-deoxyuridine, it has attracted interest as a modulator of fluoropyrimidine chemotherapy [3,7]. Understanding its catalytic mechanism, regulation, and role in disease is therefore relevant for cancer biology, antimicrobial research, and metabolic engineering.
deoxyuridine phosphorylase activity At A Glance
| GO ID | GO:0047847 |
|---|---|
| GO term | deoxyuridine phosphorylase activity |
| Ontology | molecular_function |
| Synonym | None listed in QuickGO |
| Definition | Catalysis of the reaction: 2'-deoxyuridine + phosphate = 2-deoxy-alpha-D-ribose 1-phosphate + uracil |
| Major function | Phosphorolysis of deoxyuridine to uracil and deoxyribose-1-phosphate |
| Related activity | Pyrimidine nucleoside phosphorylase; also acts on 5-fluoro-2'-deoxyuridine |
| Inhibitors | 5-Azauracil inhibits both uridine and deoxyuridine phosphorylase activities in mouse liver extracts |
| Tissue distribution | Detected in regenerating rat liver, human lymphoblasts, and Mycoplasma mycoides [2,5,6] |
What Is GO:0047847?
According to the Gene Ontology, deoxyuridine phosphorylase activity (GO:0047847) is defined as the catalysis of the reaction: 2'-deoxyuridine + phosphate = 2-deoxy-alpha-D-ribose 1-phosphate + uracil. In other words, the enzyme breaks the glycosidic bond of deoxyuridine using inorganic phosphate, releasing uracil and a phosphorylated deoxyribose moiety. This activity is a type of pentosyltransferase and is involved in pyrimidine salvage and catabolism.
Why Is deoxyuridine phosphorylase activity Important in Cell Biology?
Deoxyuridine phosphorylase activity is important because it controls the cellular levels of deoxyuridine and its phosphorylated derivatives, which are precursors for DNA synthesis and repair. By cleaving deoxyuridine, the enzyme provides uracil and deoxyribose-1-phosphate for salvage or catabolism, influencing nucleotide pool balance. In cancer therapy, the enzyme can activate or inactivate fluoropyrimidine drugs such as 5-fluoro-2'-deoxyuridine, thereby affecting drug sensitivity [3,7]. In microorganisms like Mycoplasma mycoides, pyrimidine deoxyribonucleotide metabolism enzymes, including deoxyuridine phosphorylase, are essential for survival and proliferation. Thus, this activity is a potential target for antimicrobial and anticancer strategies.
• Regulates deoxyuridine availability for DNA synthesis and repair.
• Modulates the cytotoxicity of 5-fluoro-2'-deoxyuridine and other fluoropyrimidines [3,7].
• Inhibited by 5-azauracil, linking it to pyrimidine analog chemotherapy.
• Shows increased activity in regenerating rat liver, suggesting a role in proliferation.
• Present in human lymphoblasts and affected by methotrexate treatment.
• Found in Mycoplasma mycoides, where it contributes to pyrimidine salvage.
• Human tissues degrade uridine much less than thymidine, indicating distinct roles for deoxyuridine phosphorylase.
• Potential target for irreversible inhibitors designed against tumor-derived enzyme.
• Contributes to thymineless death in Escherichia coli mutants with defective pyrimidine metabolism.
• Relevant for metabolic engineering of nucleoside analog production.
What Happens During deoxyuridine phosphorylase activity?
Substrate binding and phosphorolysis
In simple terms: The enzyme grabs deoxyuridine and a phosphate molecule, then splits deoxyuridine into two pieces.
Deoxyuridine phosphorylase binds 2'-deoxyuridine and inorganic phosphate in its active site. The enzyme catalyzes the cleavage of the N-glycosidic bond between the deoxyribose and uracil moieties, yielding 2-deoxy-alpha-D-ribose 1-phosphate and free uracil. This phosphorolytic reaction is reversible and is a key step in pyrimidine salvage.
Specificity for deoxyuridine and analogs
In simple terms: The enzyme prefers deoxyuridine but can also act on similar molecules like 5-fluoro-2'-deoxyuridine.
Pyrimidine nucleoside phosphorylases exhibit distinct specificities. Deoxyuridine phosphorylase activity is specific for deoxyuridine and can also phosphorolyze 5-fluoro-2'-deoxyuridine, as shown in studies on Walker 256 rat tumor enzyme [3,7]. This broad specificity has implications for drug metabolism.
Inhibition by pyrimidine analogs
In simple terms: Certain drugs can block the enzyme, slowing down the reaction.
5-Azauracil inhibits both uridine phosphorylase and deoxyuridine phosphorylase activities in cell-free extracts of mouse liver. This inhibition can alter deoxyuridine metabolism and has been studied in the context of chemotherapy.
Role in pyrimidine salvage and catabolism
In simple terms: The reaction helps cells recycle or break down deoxyuridine.
The products of deoxyuridine phosphorylase activity, uracil and deoxyribose-1-phosphate, can enter salvage pathways or be further catabolized. In cultured human lymphoblasts treated with methotrexate, deoxyuridine metabolism is perturbed, highlighting the enzyme's role in nucleotide pool regulation. In Mycoplasma mycoides, enzymes of pyrimidine deoxyribonucleotide metabolism, including deoxyuridine phosphorylase, are essential for growth.
Physiological regulation and tissue distribution
In simple terms: The enzyme's activity changes depending on the tissue and growth state.
Deoxyuridine phosphorylase activity increases in regenerating rat liver after uridine administration, suggesting regulation linked to proliferation. Human tissues degrade uridine much less than thymidine, indicating tissue-specific differences in pyrimidine nucleoside phosphorylase activities. These variations may influence drug responses.
Key Genes Involved in GO:0047847 deoxyuridine phosphorylase activity
The following genes and proteins are directly or indirectly associated with deoxyuridine phosphorylase activity (GO:0047847) based on published biochemical and genetic studies.
| Gene | Major Role | Research Relevance |
|---|---|---|
| UPP1 | Uridine phosphorylase 1; can also act on deoxyuridine | Inhibited by 5-azauracil; involved in pyrimidine salvage |
| UPP2 | Uridine phosphorylase 2; tissue-specific | May contribute to deoxyuridine phosphorylase activity in some tissues |
| TYMP | Thymidine phosphorylase; also acts on deoxyuridine | Shares substrate overlap; studied in fluoropyrimidine metabolism |
| DPYD | Dihydropyrimidine dehydrogenase; catabolizes uracil and thymine | Downstream of deoxyuridine phosphorylase in pyrimidine catabolism |
| TYMS | Thymidylate synthase; de novo dTMP synthesis | Affected by deoxyuridine pool changes |
| DHFR | Dihydrofolate reductase; folate metabolism | Methotrexate treatment alters deoxyuridine metabolism |
| MTHFR | Methylenetetrahydrofolate reductase | Folate cycle intersects with pyrimidine synthesis |
| UCK1 | Uridine-cytidine kinase 1 | Phosphorylates uridine and cytidine; salvage pathway |
| UCK2 | Uridine-cytidine kinase 2 | Mitochondrial salvage of pyrimidines |
| CMPK1 | UMP-CMP kinase | Phosphorylates UMP and CMP in salvage |
| NME1 | Nucleoside diphosphate kinase 1 | Maintains nucleotide pools |
| NME2 | Nucleoside diphosphate kinase 2 | Maintains nucleotide pools |
| RRM1 | Ribonucleotide reductase subunit M1 | Provides deoxyribonucleotides for DNA synthesis |
| RRM2 | Ribonucleotide reductase subunit M2 | Provides deoxyribonucleotides for DNA synthesis |
| DUT | Deoxyuridine triphosphatase | Prevents uracil incorporation into DNA |
| UNG | Uracil-DNA glycosylase | Removes uracil from DNA |
| TK1 | Thymidine kinase 1 | Salvage of thymidine; pyrimidine metabolism |
| TK2 | Thymidine kinase 2 | Mitochondrial thymidine salvage |
How Is deoxyuridine phosphorylase activity Regulated?
Deoxyuridine phosphorylase activity is regulated at multiple levels. In regenerating rat liver, uridine administration affects the activities of both uridine and deoxyuridine phosphorylase, suggesting nutritional and hormonal regulation. In cultured human lymphoblasts, methotrexate treatment alters deoxyuridine metabolism, indicating that folate pathway inhibition can influence enzyme activity or substrate availability. Additionally, tissue-specific differences in uridine versus thymidine degradation imply differential expression or regulation of pyrimidine nucleoside phosphorylases. However, specific transcriptional or post-translational regulators of deoxyuridine phosphorylase remain to be fully defined.
deoxyuridine phosphorylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| TYMP | Fluoropyrimidine sensitivity in cancer | Knockout in cancer cell lines; drug response assays |
| UPP1 | Pyrimidine salvage in tumors | Overexpression and knockout in HeLa or MCF7 cells |
| DPYD | 5-Fluorouracil toxicity | Patient-derived organoids with point mutations |
| DUT | DNA repair deficiency and thymineless death | CRISPR knockout in E. coli and human cells |
| MTHFR | Methotrexate response in leukemia | Knock-in of variant alleles in lymphoblasts |
Cancer and chemotherapy response
Deoxyuridine phosphorylase activity can modulate the efficacy of fluoropyrimidine drugs. The enzyme phosphorolyzes 5-fluoro-2'-deoxyuridine, potentially affecting its activation or degradation. Irreversible inhibitors targeting 5-fluoro-2'-deoxyuridine phosphorylase from Walker 256 rat tumor have been developed, highlighting its potential as a therapeutic target. In human lymphoblasts, methotrexate treatment perturbs deoxyuridine metabolism, which may influence drug sensitivity.
Microbial infections
Mycoplasma mycoides subsp. mycoides possesses enzymes of pyrimidine deoxyribonucleotide metabolism, including deoxyuridine phosphorylase, which are essential for its growth. Inhibiting this activity could provide a strategy against mycoplasma infections.
Thymineless death and DNA damage
In Escherichia coli mutants with defective pyrimidine metabolism, virtually thymineless DNA synthesis leads to cell death, a process in which deoxyuridine phosphorylase may influence deoxyuridine availability. This links the enzyme to DNA repair and genome stability.
From deoxyuridine phosphorylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of deoxyuridine phosphorylase alter deoxyuridine levels? | CRISPR knockout of UPP1/TYMP in human cell lines |
| Does a point mutation in the active site abolish catalysis? | Point-mutation knock-in of catalytic residues |
| Can a tagged enzyme be used for localization studies? | Knock-in of FLAG- or GFP-tagged deoxyuridine phosphorylase |
| Does overexpression sensitize cells to fluoropyrimidines? | Overexpression of UPP1 or TYMP in cancer cells |
| Which genes interact with deoxyuridine phosphorylase in pyrimidine metabolism? | CRISPR library screening with metabolic readouts |
| How does methotrexate affect deoxyuridine metabolism? | Knockout of DHFR or MTHFR in lymphoblast models |
How to Study the deoxyuridine phosphorylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric phosphorylase assay | Uracil formation from deoxyuridine | Enzyme kinetics and inhibitor screening |
| HPLC-based assay | Substrate depletion and product formation | Specificity for 5-fluoro-2'-deoxyuridine |
| LC-MS/MS metabolomics | Intracellular deoxyuridine and nucleotide pools | Methotrexate effects in lymphoblasts |
| CRISPR knockout screening | Gene essentiality and synthetic lethality | Pyrimidine metabolism networks |
| Enzyme inhibitor profiling | IC50 values for analogs | Drug development against tumor enzyme |
| Tissue activity assays | Enzyme activity in tissue extracts | Comparison of uridine vs thymidine degradation |
| Microbial growth assays | Mycoplasma proliferation | Target validation in M. mycoides |
| Regenerating liver model | Enzyme activity changes after partial hepatectomy | Proliferation-linked regulation |
Enzymatic assays for deoxyuridine phosphorylase activity
Classic biochemical assays measure the conversion of 2'-deoxyuridine to uracil using spectrophotometric or radiometric methods. Inhibition by 5-azauracil can be assessed in cell-free extracts. Specificity for 5-fluoro-2'-deoxyuridine can be tested using HPLC-based assays.
Metabolic profiling and nucleotide pool analysis
LC-MS/MS can quantify deoxyuridine, uracil, and nucleotide pools in cells with altered deoxyuridine phosphorylase activity. Such approaches have been used to study methotrexate-treated lymphoblasts and to compare uridine versus thymidine degradation in human tissues.
Genetic screens and CRISPR libraries
CRISPR knockout libraries targeting pyrimidine metabolism genes can identify synthetic lethal interactions with deoxyuridine phosphorylase. This is particularly relevant for understanding thymineless death in E. coli mutants and for identifying drug targets in Mycoplasma.
Structural and inhibitor studies
X-ray crystallography and computational docking can reveal how inhibitors like 5-azauracil or benzyluracil derivatives bind to the enzyme [1,7]. Irreversible inhibitors designed against 5-fluoro-2'-deoxyuridine phosphorylase provide templates for drug development.
How CRISPR Can Be Used to Study GO:0047847 deoxyuridine phosphorylase activity
Knockout
CRISPR knockout of UPP1, UPP2, or TYMP can abolish deoxyuridine phosphorylase activity in cell lines, enabling studies of pyrimidine salvage and drug sensitivity. Such models are useful for testing fluoropyrimidine cytotoxicity and for metabolic profiling.
Point Mutation
Point mutations in catalytic residues of deoxyuridine phosphorylase can be introduced via CRISPR base editing or HDR to dissect mechanism. These models help determine which residues are essential for phosphorolysis and inhibitor binding [1,7].
Knock-in
Knock-in of tagged versions (e.g., FLAG, GFP) of the enzyme allows localization and interaction studies. Knock-in of disease-associated variants can model altered enzyme activity in cancer or metabolic disorders [2,8].
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of deoxyuridine phosphorylase can sensitize or desensitize cells to nucleoside analogs. Overexpression models are valuable for studying drug metabolism and resistance [3,7].
How EDITGENE Supports deoxyuridine phosphorylase activity Research
Researchers studying deoxyuridine phosphorylase activity-related genes often need to determine whether a candidate gene is causally involved in pyrimidine metabolism, drug response, or disease. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for deoxyuridine phosphorylase activity research.
Frequently Asked Questions About deoxyuridine phosphorylase activity
What is deoxyuridine phosphorylase activity?
It is a molecular function (GO:0047847) that catalyzes the conversion of 2'-deoxyuridine and phosphate to 2-deoxy-alpha-D-ribose 1-phosphate and uracil.
What genes are involved in deoxyuridine phosphorylase activity?
Genes such as UPP1, UPP2, and TYMP encode enzymes with deoxyuridine phosphorylase activity or overlapping specificity [1,3].
How is deoxyuridine phosphorylase activity regulated?
It can be regulated by tissue-specific expression, proliferation signals, and folate pathway inhibition, as seen in regenerating liver and methotrexate-treated cells [2,5].
What diseases are associated with deoxyuridine phosphorylase activity?
It is linked to cancer chemotherapy response, microbial infections, and thymineless death in bacteria [3,4,6].
What is the reaction catalyzed by deoxyuridine phosphorylase?
2'-Deoxyuridine + phosphate = 2-deoxy-alpha-D-ribose 1-phosphate + uracil.
Is deoxyuridine phosphorylase inhibited by 5-azauracil?
Yes, 5-azauracil inhibits both uridine and deoxyuridine phosphorylase activities in mouse liver extracts.
Can deoxyuridine phosphorylase act on 5-fluoro-2'-deoxyuridine?
Yes, it can phosphorolyze 5-fluoro-2'-deoxyuridine, which is relevant for fluoropyrimidine drug metabolism.
How can I study deoxyuridine phosphorylase activity in the lab?
Biochemical assays, LC-MS metabolomics, and CRISPR knockout models are commonly used [1,2,4].
What cell models are available for deoxyuridine phosphorylase research?
EDITGENE offers knockout, point-mutation, knock-in, and overexpression models for genes like UPP1 and TYMP.
Why is deoxyuridine phosphorylase important for cancer therapy?
It modulates the activation and degradation of fluoropyrimidine drugs, influencing chemotherapy efficacy [3,7].
Conclusion
Deoxyuridine phosphorylase activity (GO:0047847) is a central enzymatic function in pyrimidine salvage and catabolism, with direct implications for nucleotide pool regulation, drug metabolism, and microbial growth. Its ability to act on deoxyuridine and fluorinated analogs makes it a target of interest in cancer and infectious disease research. By leveraging CRISPR-based knockout, point-mutation, knock-in, and overexpression models, researchers can dissect its precise roles and identify new therapeutic opportunities.
References
- 1. CIHAK A et al.. 1964. INHIBITION BY 5-AZAURACIL OF THE URIDINE PHOSPHORYLASE AND DEOXYURIDINE PHOSPHORYLASE ACTIVITIES IN CELL-FREE EXTRACT OF MOUSE LIVER.. Biochim Biophys Acta 80:672-4 PMID: 14156740
- 2. Perez DJ et al.. 1984. Deoxyuridine metabolism in cultured human lymphoblasts treated with methotrexate.. Cancer Res 44(2):457-60 PMID: 6198074
- 3. Woodman PW et al.. 1980. Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2'-deoxyuridine.. Cancer Res 40(3):507-11 PMID: 6451286
- 4. el-Hajj HH et al.. 1992. Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth.. J Bacteriol 174(13):4450-6 PMID: 1624437
- 5. YAMADA EW. 1962. The effect of uridine administration on the activities of uridine and deoxyuridine phosphorylase of regenerating rat liver.. Biochem Biophys Res Commun 8:232-7 PMID: 14038100
- 6. Neale GA et al.. 1983. Enzymes of pyrimidine deoxyribonucleotide metabolism in Mycoplasma mycoides subsp. mycoides.. J Bacteriol 156(3):1001-5 PMID: 6139361
- 7. Kelley JL et al.. 1982. Irreversible enzyme inhibitors. 202. Candidate active-site-directed irreversible inhibitors of 5-fluoro-2'-deoxyuridine phosphorylase from Walker 256 rat tumor derived from 1-benzyl-5-(3-ethoxybenzyl)uracil.. J Med Chem 25(5):600-3 PMID: 6211547
- 8. Liermann B et al.. 1984. Human tissues degrade uridine much less than thymidine. Possible consequence for 5-fluorouracil therapy.. Biochem Pharmacol 33(5):721-4 PMID: 6712705
