GO:0004157 dihydropyrimidinase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004157 dihydropyrimidinase activity catalyzes the hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate, the second step of pyrimidine degradation.
• The enzyme is also known as D-hydantoinase and participates in the reductive pyrimidine catabolic pathway that clears uracil and thymine.
• Dihydropyrimidinase protects cells from DNA replication stress caused by cytotoxic pyrimidine metabolites.
• Pathogenic variants in the human DPYS gene alter enzyme structure and are linked to dihydropyrimidinuria, a metabolic disorder.
• Dihydropyrimidinase activity is found across species, from plants to yeast to humans, and can hydrolyze polyamides in industrial contexts.
• Studying this activity requires enzyme assays, structural modeling, and CRISPR-based cell models to dissect its role in metabolism and disease.
Description
Dihydropyrimidinase activity (GO:0004157) is a molecular function that catalyzes the hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate, a key step in the reductive pyrimidine degradation pathway. This enzymatic activity is essential for the breakdown of uracil and thymine, and its dysfunction can lead to the accumulation of cytotoxic metabolites that interfere with DNA replication. Researchers study dihydropyrimidinase to understand pyrimidine metabolism, drug response to fluoropyrimidines, and inherited metabolic disorders. The enzyme is conserved across evolution, with homologs identified in plants, yeast, and humans, and it has attracted attention for its ability to hydrolyze diverse substrates, including hydantoins and polyamides. In clinical contexts, dihydropyrimidinase activity is relevant to cancer chemotherapy, as it influences the catabolism of fluoropyrimidine drugs. Moreover, recent structural and functional studies have revealed how pathogenic variants in the human DPYS gene impair enzyme function, providing insights into disease mechanisms. This article synthesizes authoritative QuickGO data and verified PubMed literature to provide a comprehensive overview of dihydropyrimidinase activity, its genes, regulation, disease associations, and research methodologies.
dihydropyrimidinase activity At A Glance
| GO ID | GO:0004157 |
|---|---|
| GO term | dihydropyrimidinase activity |
| Ontology | molecular_function |
| Synonym | 5,6-dihydropyrimidine amidohydrolase activity; D-hydantoinase activity; hydantoinase activity; hydantoin peptidase activity; hydropyrimidine hydrase activity; pyrimidine hydrase activity |
| Major function | Catalyzes the hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate |
| Reaction | 5,6-dihydrouracil + H2O = 3-ureidopropionate |
| Pathway | Reductive pyrimidine degradation |
| Substrates | 5,6-dihydrouracil, dihydrothymine, hydantoins |
| Cofactors | Zinc ion (typically) |
What Is GO:0004157?
Dihydropyrimidinase activity (GO:0004157) is defined as the catalysis of the reaction: 5,6-dihydrouracil + H2O = 3-ureidopropionate. This activity is synonymous with 5,6-dihydropyrimidine amidohydrolase, D-hydantoinase, hydantoinase, hydantoin peptidase, hydropyrimidine hydrase, and pyrimidine hydrase. It belongs to the molecular_function ontology and is a central enzyme in the reductive pyrimidine catabolic pathway.
Why Is dihydropyrimidinase activity Important in Cell Biology?
Dihydropyrimidinase activity is critical for maintaining pyrimidine homeostasis and preventing the accumulation of cytotoxic intermediates that can cause DNA replication stress. It is also a key enzyme in the metabolism of fluoropyrimidine drugs, influencing their efficacy and toxicity in cancer therapy. Genetic defects in dihydropyrimidinase lead to dihydropyrimidinuria, a metabolic disorder with neurological symptoms. Furthermore, the enzyme's ability to hydrolyze hydantoins and polyamides has biotechnological applications. Understanding its regulation and structure is essential for developing therapeutic strategies and for interpreting pharmacogenomic data.
• Maintains pyrimidine homeostasis by degrading uracil and thymine.
• Prevents DNA replication stress from cytotoxic metabolites.
• Influences fluoropyrimidine drug metabolism and toxicity.
• Mutations cause dihydropyrimidinuria, a rare metabolic disorder.
• Serves as a target for enzyme inhibitors like plumbagin.
• Has industrial potential for polyamide hydrolysis.
• Conserved in plants, yeast, and humans, aiding evolutionary studies.
• Provides a model for studying protein misfolding and structural variants.
• Contributes to neuroendocrine prostate cancer lineage plasticity via DPYSL5, a related protein.
• Relevant to viral infections, as RNA profiling shows metabolic shifts.
Molecular Mechanism of dihydropyrimidinase activity
Substrate Recognition and Binding
In simple terms: The enzyme grabs the substrate molecule and holds it in place for chemical reaction.
Dihydropyrimidinase binds 5,6-dihydrouracil or dihydrothymine in its active site, positioning the substrate for hydrolysis. Structural studies of pathogenic variants reveal that specific residues are critical for substrate binding and catalysis. The enzyme also accepts hydantoins, reflecting its broad substrate specificity.
Catalytic Hydrolysis
In simple terms: A water molecule splits the substrate into a smaller product.
The catalytic mechanism involves a water molecule attacking the carbonyl carbon of the dihydropyrimidine ring, leading to ring opening and formation of 3-ureidopropionate. This reaction is dependent on the presence of a zinc ion in the active site, which activates the water molecule.
Cofactors and Metal Dependence
In simple terms: A metal helper in the enzyme makes the reaction faster.
Dihydropyrimidinase is a metalloenzyme that typically requires a zinc ion for activity. The zinc ion coordinates with histidine and aspartate residues to stabilize the transition state. Mutations that affect metal binding can abolish enzyme activity.
Regulation of Enzyme Activity
In simple terms: The enzyme's speed can be turned up or down by other molecules.
Dihydropyrimidinase activity can be inhibited by compounds such as plumbagin, which was isolated from Nepenthes miranda extract. Additionally, the enzyme's expression and activity may be regulated at the transcriptional level in response to metabolic demands, although specific regulators are not fully defined.
Structural Insights from Pathogenic Variants
In simple terms: Changes in the enzyme's shape can break its function.
Molecular dynamics simulations of pathogenic variants in dihydropyrimidinase have shown that single amino acid substitutions can cause structural destabilization, reduced substrate affinity, or impaired catalysis. These findings help explain the molecular basis of dihydropyrimidinuria.
Key Genes Involved in GO:0004157 dihydropyrimidinase activity
The following genes and proteins are directly or indirectly associated with dihydropyrimidinase activity and pyrimidine metabolism.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DPYS | Encodes human dihydropyrimidinase, catalyzing the second step of pyrimidine degradation | Mutations cause dihydropyrimidinuria; target for structural and functional studies |
| DPYD | Encodes dihydropyrimidine dehydrogenase, the first enzyme in pyrimidine catabolism | Pharmacogenomic marker for fluoropyrimidine toxicity |
| UPB1 | Encodes beta-ureidopropionase, the third enzyme in pyrimidine degradation | Deficiency leads to metabolic disorders; studied alongside DPYS |
| DPYSL5 | Encodes dihydropyrimidinase-like 5, involved in neuronal development | Highly expressed in neuroendocrine prostate cancer; promotes lineage plasticity |
| DPYSL2 | Encodes dihydropyrimidinase-like 2 (CRMP2), involved in axon guidance | Related to cytoskeletal dynamics; not directly GO:0004157 but shares homology |
| DPYSL3 | Encodes dihydropyrimidinase-like 3 (CRMP4) | Implicated in neuronal differentiation and cancer |
| DPYSL4 | Encodes dihydropyrimidinase-like 4 (CRMP3) | Studied in neurodevelopment |
| DPYS (yeast) | Saccharomyces kluyveri dihydropyrimidinase | Hydrolyzes polyamides; biotechnological applications |
| DPYS (plant) | Pea plant dihydropyrimidinase | Early evidence of enzyme activity in plants |
| Nepenthes miranda hydantoinase | Plant-derived enzyme inhibited by plumbagin | Source of natural inhibitors |
| TBEV proteins | Viral effectors that may alter pyrimidine metabolism | RNA profiling of infected neurons shows metabolic shifts |
| EZH2 | Epigenetic regulator interacting with DPYSL5 | Modulates lineage plasticity in prostate cancer |
| PRC2 | Polycomb repressive complex 2, interacts with DPYSL5 | Epigenetic silencing in cancer |
| Zinc ion | Cofactor for dihydropyrimidinase | Essential for catalysis |
| 5,6-dihydrouracil | Substrate for dihydropyrimidinase | Key metabolite in pyrimidine degradation |
| 3-ureidopropionate | Product of dihydropyrimidinase reaction | Biomarker for enzyme activity |
| Plumbagin | Inhibitor of dihydropyrimidinase | Natural compound from Nepenthes miranda |
| Hydantoin | Alternative substrate | Used to assay D-hydantoinase activity |
How Is dihydropyrimidinase activity Regulated?
Dihydropyrimidinase activity is primarily regulated at the level of gene expression and through post-translational modifications, although specific mechanisms remain incompletely understood. The enzyme's activity can be inhibited by small molecules such as plumbagin, which binds to the active site and blocks catalysis. In cancer cells, DPYSL5, a related protein, is regulated by EZH2/PRC2, suggesting epigenetic control of dihydropyrimidinase-like proteins. Additionally, metabolic flux through the pyrimidine degradation pathway may be influenced by substrate availability and feedback inhibition by downstream products.
dihydropyrimidinase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DPYS | Dihydropyrimidinuria | Knockout cell lines (e.g., HEK293) and patient-derived fibroblasts |
| DPYD | Fluoropyrimidine toxicity | Isogenic cell lines with point mutations |
| DPYSL5 | Neuroendocrine prostate cancer | Overexpression and knockout in prostate cancer cell lines |
| DPYS (yeast) | Polyamide hydrolysis | Yeast knockout and overexpression |
| DPYS (plant) | Plant metabolism | Plant knockout models |
Dihydropyrimidinuria
Dihydropyrimidinuria is an inherited metabolic disorder caused by mutations in the DPYS gene, leading to reduced dihydropyrimidinase activity. Patients exhibit elevated levels of dihydrouracil and dihydrothymine in urine and may present with neurological symptoms, including seizures and developmental delay. Structural studies of pathogenic variants provide insights into the molecular basis of the disease.
Cancer and Chemotherapy Response
Dihydropyrimidinase activity is part of the pyrimidine catabolic pathway that metabolizes fluoropyrimidine drugs such as 5-fluorouracil. Altered enzyme activity can affect drug clearance and toxicity, making it a pharmacogenomic factor. Additionally, DPYSL5, a related protein, is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.
DNA Replication Stress
Loss of dihydropyrimidinase activity leads to the accumulation of cytotoxic metabolites that cause DNA replication stress. This can trigger cell cycle arrest and apoptosis, highlighting the enzyme's protective role in genome stability.
Viral Infections
Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals changes in metabolic pathways, including pyrimidine metabolism, suggesting that viral infection may alter dihydropyrimidinase activity.
From dihydropyrimidinase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does DPYS loss cause metabolite accumulation? | DPYS knockout cell line (e.g., HAP1) |
| How do pathogenic variants affect enzyme structure? | Point-mutation knock-in of DPYS variants |
| Can we tag DPYS for localization studies? | Knock-in of fluorescent tag (e.g., GFP) |
| Does DPYSL5 overexpression drive lineage plasticity? | Overexpression in prostate cancer cells |
| Is dihydropyrimidinase activity inhibited by plumbagin? | Enzyme assay with purified protein |
| Does viral infection alter pyrimidine metabolism? | TBEV-infected neuron cultures |
How to Study the dihydropyrimidinase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme activity assay | Conversion of substrate to product | Screening inhibitors, kinetic studies |
| Molecular dynamics simulation | Protein structural stability and dynamics | Assessing pathogenic variants |
| CRISPR knockout | Loss of gene function | Studying metabolic consequences |
| CRISPR point mutation | Specific amino acid changes | Modeling inherited disorders |
| CRISPR knock-in | Tagged or reporter gene | Localization and interaction studies |
| RNA-seq | Transcriptome changes | Pathway analysis in infection |
| Metabolomics | Metabolite levels | Quantifying dihydrouracil and 3-ureidopropionate |
Enzyme Activity Assays
Dihydropyrimidinase activity is typically measured using spectrophotometric or chromatographic assays that monitor the conversion of 5,6-dihydrouracil to 3-ureidopropionate. These assays can be performed with purified enzyme or cell lysates and are useful for screening inhibitors.
Structural Biology and Molecular Dynamics
X-ray crystallography and molecular dynamics simulations provide insights into the enzyme's active site and the impact of pathogenic variants. These methods help rationalize loss-of-function mutations and guide drug design.
CRISPR-Cas9 Genome Editing
CRISPR-Cas9 is used to generate knockout, point-mutation, and knock-in cell models to study dihydropyrimidinase function in a cellular context. These models enable precise interrogation of the enzyme's role in metabolism and disease.
RNA Profiling and Metabolomics
RNA sequencing and metabolomics can reveal changes in pyrimidine metabolism upon viral infection or genetic manipulation. Integrative analyses link gene expression to metabolite levels, providing a systems-level view.
How CRISPR Can Be Used to Study GO:0004157 dihydropyrimidinase activity
Knockout
CRISPR knockout of DPYS in cell lines such as HAP1 or HEK293 abolishes dihydropyrimidinase activity, leading to accumulation of 5,6-dihydrouracil and increased DNA replication stress. These models are valuable for studying the metabolic and cellular consequences of enzyme loss.
Point Mutation
Introducing specific pathogenic variants (e.g., missense mutations found in dihydropyrimidinuria patients) via CRISPR point mutation allows researchers to assess the structural and functional impact of these mutations in a physiological context.
Knock-in
Knock-in of a fluorescent tag (e.g., GFP) at the endogenous DPYS locus enables real-time imaging of enzyme localization and dynamics. This approach can also be used to introduce epitope tags for proteomic studies.
Overexpression
Overexpression of wild-type or mutant DPYS using CRISPR activation or lentiviral vectors can model conditions of enzyme excess and test gain-of-function effects. This is particularly useful for studying DPYSL5 in cancer lineage plasticity.
How EDITGENE Supports dihydropyrimidinase activity Research
Researchers studying dihydropyrimidinase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic pathways, disease progression, or drug response. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models, enabling functional validation and mechanistic studies.
Contact EDITGENE today to design your custom CRISPR model for dihydropyrimidinase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| DPYSL5 Knockout HEK293 Cell Line | EDJ-KQ2522 | Human | 56896 | Details Get a Quote |
| DPYSL2 Knockout HEK293 Cell Line | EDJ-KQ3136 | Human | 1808 | Details Get a Quote |
| CRMP1 Knockout HEK293 Cell Line | EDJ-KQ4347 | Human | 1400 | Details Get a Quote |
| DPYS Knockout HEK293 Cell Line | EDJ-KQ4473 | Human | 1807 | Details Get a Quote |
| DPYSL3 Knockout HEK293 Cell Line | EDJ-KQ4475 | Human | 1809 | Details Get a Quote |
| DPYSL4 Knockout HEK293 Cell Line | EDJ-KQ7094 | Human | 10570 | Details Get a Quote |
| DPYSL5 Knockout A-549 Cell Line | EDJ-KQ23135 | Human | 56896 | Details Get a Quote |
| DPYSL5 Knockout HCT 116 Cell Line | EDJ-KQ23136 | Human | 56896 | Details Get a Quote |
| DPYSL3 Knockout HCT 116 Cell Line | EDJ-KQ27039 | Human | 1809 | Details Get a Quote |
| DPYSL3 Knockout HeLa Cell Line | EDJ-KQ27040 | Human | 1809 | Details Get a Quote |
| DPYSL4 Knockout A-549 Cell Line | EDJ-KQ31939 | Human | 10570 | Details Get a Quote |
| DPYSL4 Knockout HCT 116 Cell Line | EDJ-KQ31940 | Human | 10570 | Details Get a Quote |
| DPYSL2 Knockout A-549 Cell Line | EDJ-KQ24513 | Human | 1808 | Details Get a Quote |
| DPYSL2 Knockout HCT 116 Cell Line | EDJ-KQ24514 | Human | 1808 | Details Get a Quote |
| DPYSL2 Knockout HeLa Cell Line | EDJ-KQ24515 | Human | 1808 | Details Get a Quote |
Displaying Records 1 To 15 Of 24 Records
Frequently Asked Questions About dihydropyrimidinase activity
What is dihydropyrimidinase activity?
Dihydropyrimidinase activity (GO:0004157) is the catalysis of the reaction: 5,6-dihydrouracil + H2O = 3-ureidopropionate, a key step in pyrimidine degradation.
What genes are involved in dihydropyrimidinase activity?
The primary gene is DPYS, which encodes the enzyme dihydropyrimidinase. Related genes include DPYD, UPB1, and DPYSL family members.
What diseases are associated with dihydropyrimidinase deficiency?
Dihydropyrimidinuria, a metabolic disorder with neurological symptoms, is caused by mutations in DPYS.
How is dihydropyrimidinase activity measured?
It is measured using enzyme assays that detect the conversion of 5,6-dihydrouracil to 3-ureidopropionate, often with spectrophotometry or HPLC.
What is the role of dihydropyrimidinase in cancer?
It metabolizes fluoropyrimidine drugs and protects against DNA replication stress; altered activity affects chemotherapy response.
Can dihydropyrimidinase be inhibited?
Yes, compounds like plumbagin inhibit dihydropyrimidinase activity.
What is the structure of dihydropyrimidinase?
It is a zinc-dependent metalloenzyme with a TIM barrel fold; pathogenic variants disrupt its structure.
Is dihydropyrimidinase found in plants?
Yes, dihydropyrimidinase activity has been detected in pea plants.
What is the difference between DPYS and DPYSL5?
DPYS encodes the metabolic enzyme dihydropyrimidinase, while DPYSL5 encodes a related protein involved in neuronal development and cancer.
How can CRISPR help study dihydropyrimidinase?
CRISPR can create knockout, point-mutation, and knock-in models to study the enzyme's function and disease relevance.
Conclusion
Dihydropyrimidinase activity (GO:0004157) is a fundamental enzymatic function in pyrimidine catabolism with critical roles in metabolism, drug response, and disease. Understanding its mechanism, regulation, and genetic variants is essential for both basic research and clinical applications. CRISPR-based models and advanced analytical methods continue to illuminate its biology, offering new avenues for therapeutic intervention.
References
- 1. Selinger M et al.. 2022. Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.. Comput Struct Biotechnol J 20:2759-2777 PMID: 35685361
- 2. Kaarijärvi R et al.. 2024. DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.. Commun Biol 7(1):108 PMID: 38238517
- 3. Hishinuma E et al.. 2020. In Vitro Assessment of Fluoropyrimidine-Metabolizing Enzymes: Dihydropyrimidine Dehydrogenase, Dihydropyrimidinase, and β-Ureidopropionase.. J Clin Med 9(8) PMID: 32707991
- 4. Mazuś B et al.. 1965. Dihydropyrimidinase activity in pea plants.. Arkh Patol 27(8):267-73 PMID: 5869803
- 5. Huang YH et al.. 2020. Identification and characterization of dihydropyrimidinase inhibited by plumbagin isolated from Nepenthes miranda extract.. Biochimie 171-172:124-135 PMID: 32147511
- 6. Quartinello F et al.. 2023. Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides.. Front Bioeng Biotechnol 11:1158226 PMID: 37180040
- 7. Basbous J et al.. 2020. Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.. Nucleic Acids Res 48(4):1886-1904 PMID: 31853544
- 8. Kato K et al.. 2022. Structural investigation of pathogenic variants in dihydropyrimidinase using molecular dynamics simulations.. J Mol Graph Model 117:108288 PMID: 35961217
