GO:0004152 dihydroorotate dehydrogenase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004152 dihydroorotate dehydrogenase activity catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step of de novo pyrimidine biosynthesis.
• DHODH, the enzyme carrying this activity, is a mitochondrial inner-membrane protein that couples pyrimidine synthesis to the electron transport chain via ubiquinone and can use fumarate as an alternative electron acceptor.
• DHODH inhibition triggers ferroptosis and is a targetable vulnerability in cancer, and DHODH-mediated ferroptosis defence is a key mechanism of tumor survival.
• DHODH inhibitors show broad antiviral activity against mammarenaviruses, SARS-CoV-2-related viruses, and other RNA viruses, often synergizing with nucleoside analogs [2,5,6].
• Loss of DHODH or its regulators causes orotic acid accumulation, which drives liver fibrosis in MASH and is linked to inherited pyrimidine synthesis disorders.
• DHODH is a driver of macropinocytosis in tumor cells, and its inhibition reverses immunosuppression and overcomes anti-PD1 resistance.
Description
Dihydroorotate dehydrogenase (DHODH) activity, annotated as GO:0004152, is the molecular function that catalyzes the conversion of (S)-dihydroorotate to orotate, the fourth and rate-limiting step in de novo pyrimidine biosynthesis. This activity is essential for supplying pyrimidine nucleotides for DNA and RNA synthesis, and it is performed by DHODH, a flavin mononucleotide (FMN)-dependent enzyme located in the inner mitochondrial membrane. Because pyrimidine availability is critical for cell proliferation, DHODH has emerged as a therapeutic target in cancer, viral infections, and autoimmune diseases [1,2,4]. The reaction also links pyrimidine synthesis to the mitochondrial electron transport chain, as DHODH transfers electrons to ubiquinone (coenzyme Q) and, under certain conditions, to fumarate. This connection places DHODH at the interface of nucleotide metabolism, redox biology, and mitochondrial respiration [1,8]. Researchers study GO:0004152 to understand how cells balance nucleotide supply, how metabolic vulnerabilities arise in tumors, and how pharmacological inhibition of DHODH can be exploited clinically [1,4].
dihydroorotate dehydrogenase activity At A Glance
| GO ID | GO:0004152 |
|---|---|
| GO term | dihydroorotate dehydrogenase activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step of de novo pyrimidine biosynthesis. |
| Cofactor | Flavin mononucleotide (FMN); uses ubiquinone or fumarate as electron acceptor. |
| Subcellular location | Inner mitochondrial membrane, with the catalytic domain facing the intermembrane space. |
| Pathway | De novo pyrimidine biosynthesis (UMP synthesis). |
| Inhibitors | Teriflunomide, brequinar, olorofim, and other small molecules [1,7]. |
| Disease relevance | Cancer, viral infections, MASH-related liver fibrosis, and pyrimidine synthesis disorders [1,3,4]. |
What Is GO:0004152?
GO:0004152 dihydroorotate dehydrogenase activity is defined as the catalysis of the reaction: (S)-dihydroorotate + A = AH(2) + orotate, where A is an electron acceptor. In vivo, DHODH uses ubiquinone (coenzyme Q) as its primary electron acceptor, reducing it to ubiquinol, and can also use fumarate as a terminal electron acceptor under hypoxia or when the respiratory chain is impaired. This activity is a molecular_function in the Gene Ontology and is a key step in the de novo pyrimidine biosynthetic pathway.
Why Is dihydroorotate dehydrogenase activity Important in Cell Biology?
GO:0004152 is important because it controls the rate-limiting step of de novo pyrimidine synthesis, a pathway required for DNA replication, RNA transcription, and cell proliferation. DHODH inhibition selectively kills cancer cells that depend on pyrimidine synthesis and triggers ferroptosis, a form of iron-dependent cell death, making it a promising anticancer strategy. In viral infections, DHODH inhibitors block replication of mammarenaviruses and other RNA viruses, and they synergize with broad-spectrum antiviral nucleosides [2,5,6]. In liver disease, decreased DHODH activity or loss of its regulator LONP1 leads to orotic acid accumulation and exacerbates MASH-induced liver fibrosis. DHODH also drives macropinocytosis in tumor cells, and its inhibition reverses immunosuppression and overcomes anti-PD1 resistance. Finally, DHODH is a validated target in antifungal therapy, with olorofim showing potent activity against several fungal species.
• DHODH is the rate-limiting enzyme of de novo pyrimidine biosynthesis, essential for nucleotide supply in proliferating cells.
• DHODH inhibition induces ferroptosis, a non-apoptotic cell death pathway, in cancer cells.
• DHODH-mediated ferroptosis defence is a targetable vulnerability in multiple cancer types.
• DHODH inhibitors have broad antiviral activity against mammarenaviruses and other RNA viruses [2,5,6].
• DHODH inhibition synergizes with 4'-fluorouridine, a broad antiviral nucleoside.
• Loss of DHODH function or its regulator LONP1 causes orotic acid accumulation and liver fibrosis in MASH.
• DHODH drives macropinocytosis in tumor cells and contributes to immunosuppression and anti-PD1 resistance.
• Olorofim, a DHODH inhibitor, has potent in vitro activity against several fungal pathogens.
• DHODH uses fumarate as an alternative electron acceptor, linking pyrimidine synthesis to mitochondrial respiration.
• DHODH is a target for autoimmune diseases, cancer, and antiviral therapy, with several inhibitors in clinical trials [1,2,7].
What Happens During dihydroorotate dehydrogenase activity?
Substrate binding and oxidation of (S)-dihydroorotate
In simple terms: DHODH grabs dihydroorotate and removes electrons from it.
The reaction begins when (S)-dihydroorotate binds to the active site of DHODH, where the flavin mononucleotide (FMN) cofactor accepts two electrons and two protons, oxidizing dihydroorotate to orotate. This step is the only redox reaction in the de novo pyrimidine pathway and is rate-limiting for UMP synthesis.
Electron transfer to ubiquinone or fumarate
In simple terms: The electrons taken from dihydroorotate are passed to a molecule that carries them away.
Reduced FMN transfers electrons to ubiquinone (coenzyme Q) at the mitochondrial inner membrane, converting it to ubiquinol, which feeds into the respiratory chain. Under conditions where the respiratory chain is impaired or oxygen is limited, DHODH can use fumarate as a terminal electron acceptor, reducing it to succinate.
Coupling to mitochondrial respiration
In simple terms: DHODH activity is tied to how mitochondria make energy.
Because DHODH donates electrons to ubiquinone, its activity is functionally linked to complex III of the electron transport chain, and inhibition of DHODH can alter mitochondrial redox balance. This coupling explains why DHODH inhibitors can affect both pyrimidine synthesis and mitochondrial function [1,8].
Product release and pyrimidine pathway flux
In simple terms: The product orotate is released and moves to the next enzyme in the pathway.
Orotate produced by DHODH is subsequently converted to UMP by UMP synthase, completing the de novo pyrimidine pathway. The activity of DHODH therefore determines the flux of pyrimidines available for DNA and RNA synthesis, and its inhibition rapidly depletes pyrimidine pools in cells.
Key Genes Involved in GO:0004152 dihydroorotate dehydrogenase activity
The following genes and proteins are directly involved in or regulate dihydroorotate dehydrogenase activity and its associated pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DHODH | Encodes the enzyme with dihydroorotate dehydrogenase activity (GO:0004152) | Target for cancer, antiviral, and antifungal therapy [1,2,7] |
| CAD | Multienzyme complex that catalyzes the first three steps of de novo pyrimidine synthesis | Upstream regulator of DHODH substrate supply |
| UMPS | Converts orotate to UMP, the product of DHODH reaction | Downstream enzyme in pyrimidine synthesis |
| LONP1 | Mitochondrial protease that regulates DHODH expression and orotic acid levels | Loss exacerbates MASH-induced liver fibrosis |
| SLC7A11 | Cystine/glutamate antiporter that influences ferroptosis sensitivity | Modulates DHODH inhibitor-induced ferroptosis |
| GPX4 | Glutathione peroxidase that protects against lipid peroxidation | DHODH inhibition triggers ferroptosis via GPX4-dependent mechanisms |
| FSP1 | Ferroptosis suppressor protein 1, a CoQ oxidoreductase | DHODH-mediated ferroptosis defence involves CoQ and FSP1 |
| NDUFS1 | Complex I subunit of the electron transport chain | Links DHODH to mitochondrial respiration |
| SDHA | Succinate dehydrogenase, which can produce fumarate for DHODH | Provides alternative electron acceptor for DHODH |
| FH | Fumarase, interconverts fumarate and malate | Regulates fumarate availability for DHODH |
| MYC | Oncogene that drives pyrimidine synthesis and DHODH dependence | Predicts sensitivity to DHODH inhibitors |
| KRAS | Oncogene that can increase DHODH dependence in cancer | Biomarker for DHODH-targeted therapy |
| ATF4 | Stress-responsive transcription factor that can regulate DHODH expression | Links integrated stress response to pyrimidine synthesis |
| mTOR | Kinase that promotes pyrimidine synthesis and DHODH activity | Regulates cell growth and DHODH dependence |
| PD-1 | Immune checkpoint receptor whose blockade is affected by DHODH inhibition | DHODH inhibition overcomes anti-PD1 resistance |
| IFN-I | Type I interferon pathway that can be modulated by DHODH inhibitors | DHODH inhibitors show interferon-independent antiviral activity |
| CASP1 | Inflammasome caspase that can be influenced by pyrimidine metabolism | Linked to liver inflammation in MASH |
| NLRP3 | Inflammasome sensor affected by orotic acid accumulation | Potential target in MASH-related fibrosis |
How Is dihydroorotate dehydrogenase activity Regulated?
DHODH activity is regulated at multiple levels. Transcriptionally, DHODH expression is promoted by growth-promoting pathways such as mTOR and MYC, which drive pyrimidine synthesis in proliferating cells. The integrated stress response and ATF4 can also modulate DHODH expression under metabolic stress. Post-translationally, DHODH is a mitochondrial inner-membrane protein whose activity depends on its interaction with ubiquinone and the respiratory chain, and it can be regulated by the availability of fumarate as an alternative electron acceptor. The mitochondrial protease LONP1 regulates DHODH protein levels, and its loss leads to decreased DHODH expression and elevated orotic acid. Pharmacologically, DHODH is inhibited by small molecules such as teriflunomide, brequinar, and olorofim, which block pyrimidine synthesis and trigger downstream effects including ferroptosis [1,7].
dihydroorotate dehydrogenase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DHODH | Cancer, ferroptosis, anti-PD1 resistance [1,4] | DHODH knockout or point-mutation cancer cell lines; xenograft models [1,4] |
| DHODH | Viral infections (mammarenaviruses, RNA viruses) [2,5,6] | DHODH knockout cells infected with viruses; antiviral assays [2,5] |
| LONP1 | MASH-induced liver fibrosis via orotic acid | LONP1 knockout hepatocytes or liver organoids |
| DHODH | Fungal infections | Fungal DHODH knock-in in model organisms; antifungal susceptibility testing |
| DHODH | Pyrimidine synthesis disorders (orotic aciduria) | Patient-derived fibroblasts or iPSCs with DHODH mutations |
DHODH in Cancer and Ferroptosis
DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer, where DHODH inhibition induces ferroptosis and suppresses tumor growth. DHODH is also a driver of macropinocytosis in tumor cells, and its inhibition reverses immunosuppression and overcomes anti-PD1 resistance. These findings position DHODH as a promising target for cancer therapy, particularly in tumors with high pyrimidine demand [1,4].
DHODH in Viral Infections
DHODH inhibitors show broad antiviral activity against mammarenaviruses and other RNA viruses, often independent of interferon signaling. They synergize with the broad antiviral activity of 4'-fluorouridine, enhancing antiviral efficacy. Natural product inhibitors such as arzanol also inhibit human DHODH and display antiviral activity, expanding the chemical space for antiviral development.
DHODH in Liver Fibrosis and MASH
Decreased LONP1 expression exacerbates MASH-induced liver fibrosis via elevated orotic acid levels, linking DHODH dysfunction to liver disease. Orotic acid accumulation, a consequence of impaired DHODH activity, promotes fibrotic responses in the liver. This suggests that modulating DHODH activity or orotic acid levels could be a therapeutic strategy in MASH.
DHODH in Fungal Infections
The DHODH inhibitor olorofim has potent in vitro activity against Microascus/Scopulariopsis, Rasamsonia, Penicillium, and Talaromyces species, highlighting its potential as an antifungal agent. This broad-spectrum activity supports the development of DHODH inhibitors for difficult-to-treat fungal infections.
From dihydroorotate dehydrogenase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of DHODH activity cause ferroptosis sensitivity? | DHODH knockout cell lines |
| Does a specific DHODH point mutation affect enzyme activity? | DHODH point-mutation knock-in cell lines |
| Can DHODH overexpression protect against ferroptosis? | DHODH overexpression cell lines |
| How does DHODH inhibition affect antiviral response? | DHODH knockout or inhibitor-treated cells infected with viruses [2,5] |
| Does LONP1 regulate DHODH and orotic acid levels? | LONP1 knockout or overexpression liver cells |
| Does DHODH inhibition overcome anti-PD1 resistance? | DHODH knockout tumor cells in syngeneic mouse models |
How to Study the dihydroorotate dehydrogenase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| DHODH activity assay | Enzyme catalytic rate using ubiquinone or DCIP | Inhibitor screening and mutant characterization |
| LC-MS metabolomics | Orotic acid and pyrimidine intermediates | Assessing DHODH function in cells and tissues |
| Cell viability assay | Cell survival after DHODH inhibition | Cancer drug sensitivity testing |
| Lipid peroxidation assay | Ferroptosis induction | Mechanism of DHODH inhibitor-induced cell death |
| Plaque reduction assay | Viral replication inhibition [2,5] | Antiviral activity of DHODH inhibitors [2,5] |
| Broth microdilution | Fungal growth inhibition | Antifungal susceptibility testing |
| Western blot | DHODH protein expression | Regulation by LONP1 or other factors |
| CRISPR knockout screening | Gene essentiality and resistance | Identifying DHODH dependencies in cancer |
Enzymatic Activity Assays
DHODH activity can be measured spectrophotometrically by monitoring the reduction of the electron acceptor (e.g., ubiquinone or dichlorophenolindophenol) at 600 nm or by detecting orotate production. These assays are used to screen inhibitors and to characterize mutant enzymes [1,7].
Metabolomics and Orotic Acid Quantification
Liquid chromatography-mass spectrometry (LC-MS) can quantify orotic acid and other pyrimidine intermediates to assess DHODH activity in cells and tissues. Elevated orotic acid is a biomarker of impaired DHODH function in MASH and pyrimidine synthesis disorders.
Cell Viability and Ferroptosis Assays
Cell viability assays, lipid peroxidation measurements, and ferroptosis inhibitors (e.g., ferrostatin-1) are used to determine whether DHODH inhibition induces ferroptosis. These methods are essential for studying DHODH as a targetable vulnerability in cancer.
Antiviral and Antifungal Susceptibility Testing
Plaque reduction assays and viral yield assays measure the antiviral activity of DHODH inhibitors against mammarenaviruses and other viruses [2,5]. Broth microdilution assays are used to test DHODH inhibitors against fungal pathogens.
How CRISPR Can Be Used to Study GO:0004152 dihydroorotate dehydrogenase activity
Knockout
CRISPR knockout of DHODH is used to study the consequences of complete loss of dihydroorotate dehydrogenase activity, including pyrimidine auxotrophy, ferroptosis sensitivity, and effects on viral replication [1,2]. DHODH knockout cell lines are valuable tools for validating inhibitor specificity and for identifying compensatory pathways.
Point Mutation
Point mutations in DHODH can be introduced to study catalytic residues, cofactor binding, and inhibitor resistance. For example, mutations in the ubiquinone-binding site can alter electron transfer and drug sensitivity. These models help dissect the molecular mechanism of GO:0004152.
Knock-in
Knock-in of tagged DHODH (e.g., HA or GFP) allows visualization and immunoprecipitation of the enzyme to study its localization and interactions. Knock-in of disease-associated mutations can model pyrimidine synthesis disorders.
Overexpression
Overexpression of DHODH is used to test whether increased enzyme activity protects cells from ferroptosis or enhances pyrimidine supply. It can also be used to study the effects of DHODH on mitochondrial respiration and macropinocytosis [4,8].
How EDITGENE Supports dihydroorotate dehydrogenase activity Research
Researchers studying dihydroorotate dehydrogenase activity-related genes often need to determine whether a candidate gene is causally involved in pyrimidine metabolism, ferroptosis, or antiviral responses. EDITGENE provides a comprehensive suite of CRISPR-based services to create precisely engineered cell models for such studies.
Contact EDITGENE today to design your custom CRISPR model for dihydroorotate dehydrogenase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID |
|---|
Frequently Asked Questions About dihydroorotate dehydrogenase activity
What is dihydroorotate dehydrogenase activity?
Dihydroorotate dehydrogenase activity (GO:0004152) is the molecular function that catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step of de novo pyrimidine biosynthesis.
What gene encodes dihydroorotate dehydrogenase activity?
The DHODH gene encodes the enzyme dihydroorotate dehydrogenase, which carries out this activity.
What is the role of DHODH in cancer?
DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer, and DHODH inhibition can suppress tumor growth and overcome anti-PD1 resistance [1,4].
How do DHODH inhibitors work as antivirals?
DHODH inhibitors block pyrimidine synthesis, which is required for viral replication, and show broad antiviral activity against mammarenaviruses and other RNA viruses [2,5,6].
What diseases are linked to DHODH dysfunction?
DHODH dysfunction is linked to cancer, viral infections, MASH-induced liver fibrosis, and pyrimidine synthesis disorders such as orotic aciduria [1,3,4].
What is the reaction catalyzed by dihydroorotate dehydrogenase?
The reaction is (S)-dihydroorotate + A = AH(2) + orotate, where A is an electron acceptor such as ubiquinone or fumarate.
How is DHODH activity regulated?
DHODH is regulated by growth-promoting pathways like mTOR and MYC, by the integrated stress response, and by the availability of electron acceptors such as ubiquinone and fumarate [1,8].
What are the substrates and products of DHODH?
The substrate is (S)-dihydroorotate and the product is orotate; electrons are transferred to ubiquinone or fumarate.
Can DHODH be targeted for antifungal therapy?
Yes, the DHODH inhibitor olorofim has potent in vitro activity against several fungal species, including Microascus/Scopulariopsis and Rasamsonia.
What experimental models are used to study DHODH?
Common models include DHODH knockout and point-mutation cell lines, overexpression models, and CRISPR screens to study ferroptosis, antiviral responses, and cancer dependencies [1,2,4].
Conclusion
Dihydroorotate dehydrogenase activity (GO:0004152) is a critical enzymatic function in de novo pyrimidine biosynthesis, with far-reaching implications for cancer, viral infections, liver disease, and fungal infections [1,3,4,7]. Its unique position at the interface of nucleotide metabolism and mitochondrial respiration makes it a compelling target for therapeutic intervention [1,8]. Continued research using CRISPR-engineered cell models and advanced metabolomics will further elucidate its regulation and disease relevance [1,3].
References
- 1. Mao C et al.. 2021. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer.. Nature 593(7860):586-590 PMID: 33981038
- 2. Schrell L et al.. 2025. Inhibitors of dihydroorotate dehydrogenase synergize with the broad antiviral activity of 4'-fluorouridine.. Antiviral Res 233:106046 PMID: 39638153
- 3. Xu D et al.. 2026. Decreased LONP1 expression exacerbates MASH-induced liver fibrosis via elevated orotic acid levels.. J Hepatol 84(1):165-180 PMID: 40784490
- 4. Wang Y et al.. 2025. Inhibition of tumor cell macropinocytosis driver DHODH reverses immunosuppression and overcomes anti-PD1 resistance.. Immunity 58(10):2456-2471.e6 PMID: 40816268
- 5. Kim YJ et al.. 2020. Novel Dihydroorotate Dehydrogenase Inhibitors with Potent Interferon-Independent Antiviral Activity against Mammarenaviruses In Vitro.. Viruses 12(8) PMID: 32751087
- 6. Alberti M et al.. 2025. Arzanol Inhibits Human Dihydroorotate Dehydrogenase and Shows Antiviral Activity.. J Nat Prod 88(11):2586-2595 PMID: 41143446
- 7. Wiederhold NP et al.. 2023. Dihydroorotate dehydrogenase inhibitor olorofim has potent in vitro activity against Microascus/Scopulariopsis, Rasamsonia, Penicillium and Talaromyces species.. Mycoses 66(3):242-248 PMID: 36435987
- 8. Spinelli JB et al.. 2021. Fumarate is a terminal electron acceptor in the mammalian electron transport chain.. Science 374(6572):1227-1237 PMID: 34855504
