Dihydroorotate dehydrogenase (quinone), mitochondrial (DHODH) is a class 2 flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate to orotate, the fourth step and rate-limiting mitochondrial step in de novo pyrimidine biosynthesis. The enzyme is embedded in the inner mitochondrial membrane, where electrons flow from substrate to the FMN cofactor and then to ubiquinone (coenzyme Q), generating ubiquinol that feeds the respiratory chain (complex III). This directly couples pyrimidine synthesis to mitochondrial electron transport. The enzyme comprises a catalytic C-terminal TIM barrel housing FMN and substrate-binding sites, and an N-terminal membrane-associated region with two helices that anchor the protein and create a lipophilic ubiquinone-binding tunnel. Biallelic DHODH variants cause Miller syndrome (postaxial acrofacial dysostosis, MIM:263750). DHODH is a validated drug target with approved inhibitors (leflunomide/teriflunomide for autoimmune diseases) and multiple oncology programs in clinical development.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004152 dihydroorotate dehydrogenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for the generic dihydroorotate dehydrogenase activity. This is the correct enzyme activity for DHODH, though GO:0106430 (quinone-using) is more specific. Human DHODH is a class 2 enzyme that uses ubiquinone as electron acceptor (PMID:8925840, UniProt Q02127 EC 1.3.5.2). IBA annotations are phylogenetically informed and represent a good baseline. Reason: Correct core molecular function. While GO:0106430 (dihydroorotate dehydrogenase (quinone) activity) is more specific, this generic term is also accurate as DHODH does catalyze oxidation of dihydroorotate to orotate. The IBA annotation is phylogenetically sound and consistent with the literature. Supporting Evidence: PMID:8925840 Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) has been overproduced by means of a recombinant baculovirus file:human/DHODH/DHODH-uniprot.txt Catalyzes the conversion of dihydroorotate to orotate with |
| GO:0005743 mitochondrial inner membrane | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for mitochondrial inner membrane localization. This is the correct and specific localization for human DHODH. The N-terminal helices form a lipophilic ubiquinone-binding tunnel embedded in the membrane (PMID:10673429; UniProt Q02127 subcellular location). Reason: Correct core cellular component. DHODH is specifically localized to the mitochondrial inner membrane as a single-pass membrane protein with an uncleaved transit peptide required for targeting and proper membrane integration (UniProt Q02127). Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Mitochondrion inner membrane PMID:10673429 an alpha-helical domain that forms the opening of a tunnel leading to the active site. Both inhibitors share a common binding site in this tunnel |
| GO:0006207 'de novo' pyrimidine nucleobase biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for de novo pyrimidine nucleobase biosynthesis. DHODH catalyzes the fourth step in de novo pyrimidine biosynthesis, converting dihydroorotate to orotate which is subsequently converted to UMP. This is a core biological process for the enzyme. Reason: Core biological process. DHODH is essential for de novo pyrimidine biosynthesis, as demonstrated by Miller syndrome where biallelic loss-of-function mutations cause severe developmental defects due to impaired pyrimidine synthesis. Supporting Evidence: PMID:8925840 Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) file:human/DHODH/DHODH-uniprot.txt Pyrimidine metabolism; UMP biosynthesis via de novo pathway |
| GO:0009220 pyrimidine ribonucleotide biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA annotation for pyrimidine ribonucleotide biosynthesis. DHODH produces orotate which is the precursor for UMP, CTP, and other pyrimidine ribonucleotides. This is a core biological process for the enzyme. Reason: Core biological process. DHODH inhibition depletes UMP/UTP and impairs ribosome biogenesis and mRNA translation, demonstrating its essential role in pyrimidine ribonucleotide biosynthesis. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt UMP biosynthesis via de novo pathway |
| GO:0004152 dihydroorotate dehydrogenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation via combined automated methods. Correctly identifies the core enzyme activity. Duplicate of the IBA annotation but acceptable as independent computational evidence. Reason: Correct molecular function annotation. This duplicates the IBA annotation which is fine - multiple evidence codes supporting the same term strengthens confidence. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt EC=1.3.5.2 {ECO:0000269|PubMed:8925840} |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | REMOVE | Summary: IEA annotation mapping cytoplasm from InterPro. This is overly general and misleading for DHODH, which is specifically localized to the mitochondrial inner membrane, not distributed throughout the cytoplasm. Reason: This annotation is misleading. While technically mitochondria are in the cytoplasm, GO:0005737 (cytoplasm) implies cytosolic localization which is incorrect. DHODH is an integral inner mitochondrial membrane protein. The more specific term GO:0005743 (mitochondrial inner membrane) is already annotated and is the appropriate localization term. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Mitochondrion inner membrane |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | ACCEPT | Summary: IEA annotation from UniProtKB/Swiss-Prot subcellular location mapping. Correctly identifies the specific localization of DHODH. Duplicate of IBA annotation with different evidence source. Reason: Correct localization annotation. Consistent with experimental evidence from PMID:10727948 cited in UniProt for subcellular location. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Mitochondrion inner membrane |
| GO:0006207 'de novo' pyrimidine nucleobase biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: IEA annotation from InterPro mapping. Correctly identifies the core biological process. Duplicate of IBA annotation with different evidence. Reason: Correct biological process annotation with independent computational evidence supporting the IBA annotation. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Pyrimidine metabolism; UMP biosynthesis via de novo pathway |
| GO:0006221 pyrimidine nucleotide biosynthetic process | IEA GO_REF:0000043 | ACCEPT | Summary: IEA annotation from UniProt keyword mapping. This is the parent term of GO:0009220 (pyrimidine ribonucleotide biosynthetic process) and is correct but less specific. Reason: Correct but less specific than already-annotated child terms. Acceptable as part of the annotation hierarchy - provides a broader grouping. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Pyrimidine biosynthesis |
| GO:0016020 membrane | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: IEA annotation for membrane localization. Too general - more specific term GO:0005743 (mitochondrial inner membrane) is already annotated. Reason: While technically correct (DHODH is a membrane protein), this term is uninformatively general. The specific localization GO:0005743 (mitochondrial inner membrane) is already correctly annotated. This adds no additional information and clutters the annotation set. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Single-pass membrane protein |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000043 | MARK AS OVER ANNOTATED | Summary: IEA annotation for generic oxidoreductase activity. Too general - more specific terms (GO:0004152 or GO:0106430) are already annotated. Reason: This is the root term for oxidoreductase activities and is too general to be informative. The specific enzyme activity GO:0004152/GO:0106430 is already annotated. This term adds no useful information about the specific function of DHODH. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt EC=1.3.5.2 {ECO:0000269|PubMed:8925840} |
| GO:0016627 oxidoreductase activity, acting on the CH-CH group of donors | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: IEA annotation for oxidoreductase activity on CH-CH bonds. While this describes the chemistry of the reaction (dihydroorotate has a CH-CH bond that is oxidized), it is less informative than the specific enzyme activity terms already annotated. Reason: Correct but uninformatively general. The specific enzyme activity GO:0106430 (dihydroorotate dehydrogenase (quinone) activity) or GO:0004152 provides much more useful information. This intermediate-level term adds little value. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Reaction=(S)-dihydroorotate + a quinone = orotate + a quinol |
| GO:0106430 dihydroorotate dehydrogenase (quinone) activity | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for the specific quinone-using dihydroorotate dehydrogenase activity. This is THE most specific and correct molecular function term for human DHODH, distinguishing it from class 1 DHODHs that use NAD+/fumarate. Reason: This is the most specific and accurate molecular function term for human DHODH (EC 1.3.5.2). Human DHODH is a class 2 enzyme that uses ubiquinone as electron acceptor, distinguishing it from bacterial class 1 enzymes. This should be retained as a core annotation. Supporting Evidence: PMID:8925840 By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively file:human/DHODH/DHODH-uniprot.txt Reaction=(S)-dihydroorotate + a quinone = orotate + a quinol |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: IPI annotation (WITH/FROM UniProtKB:P49638, TTPA) from the HuRI large-scale binary interactome map. GO:0005515 (protein binding) is an uninformative catch-all term that gives no mechanistic insight into DHODH function. The interaction with TTPA (alpha-tocopherol transfer protein) was detected by high-throughput yeast two-hybrid and is not corroborated by an established biological role for DHODH. Reason: Per GO curation guidance, bare GO:0005515 (protein binding) is uninformative and should not be treated as a core function. The supporting evidence is a single high-throughput binary interactome screen (PMID:32296183); the TTPA interaction is not independently validated and does not describe a specific DHODH molecular function. Retained but flagged as an over-annotation rather than removed, since the underlying binary interaction may be real. Supporting Evidence: PMID:32296183 The dataset, versioned HI-III-20 (Human Interactome obtained from screening Space III, published in 2020), contains 52,569 verified PPIs involving 8,275 proteins |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: IPI annotation (WITH/FROM UniProtKB:Q6ZMZ0, RNF19B) from the HuRI large-scale binary interactome map. As with the TTPA interaction, GO:0005515 (protein binding) is an uninformative term. The RNF19B (an E3 ubiquitin-protein ligase) interaction was detected by high-throughput yeast two-hybrid without independent functional validation for DHODH. Reason: Bare GO:0005515 (protein binding) is uninformative per GO guidance and does not capture a specific DHODH molecular function. The evidence is the same single high-throughput binary interactome screen (PMID:32296183); the RNF19B interaction is unvalidated. Retained but flagged as an over-annotation rather than removed. Supporting Evidence: PMID:32296183 The dataset, versioned HI-III-20 (Human Interactome obtained from screening Space III, published in 2020), contains 52,569 verified PPIs involving 8,275 proteins |
| GO:0004151 dihydroorotase activity | IEA GO_REF:0000107 | REMOVE | Summary: INCORRECT ANNOTATION. This is a different enzyme - dihydroorotase (E.C. 3.5.2.3) catalyzes the cyclization of N-carbamoyl-L-aspartate to L-dihydroorotate, which is step 3 of pyrimidine biosynthesis. This activity is part of the CAD complex (specifically the DHO domain of CAD) in humans. DHODH (dihydroorotate dehydrogenase) catalyzes step 4, the oxidation of dihydroorotate to orotate. Reason: This is an erroneous annotation. Dihydroorotase (GO:0004151, EC 3.5.2.3) is a completely different enzyme that catalyzes the PREVIOUS step in pyrimidine biosynthesis (ring closure of carbamoyl aspartate). In humans, this activity is part of the trifunctional CAD enzyme in the cytosol. DHODH (GO:0004152, EC 1.3.5.2) catalyzes the NEXT step (oxidation of dihydroorotate). This annotation likely resulted from name similarity confusion in automated orthology transfer (GO_REF:0000107 is Ensembl Compara). Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt EC=1.3.5.2 {ECO:0000269|PubMed:8925840} PMID:8925840 Human mitochondrial dihydroorotate dehydrogenase (the fourth enzyme of pyrimidine de novo synthesis) |
| GO:0006225 UDP biosynthetic process | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: IEA annotation from Ensembl Compara orthology. DHODH produces orotate which is converted to UMP and then can be further phosphorylated to UDP. However, DHODH is not directly involved in UDP biosynthesis - it produces a precursor that is several enzymatic steps upstream. Reason: DHODH contributes to UDP biosynthesis indirectly by providing the orotate precursor. However, UDP biosynthesis requires several additional enzymatic steps after DHODH action (orotate -> OMP by UMPS -> UMP -> UDP by UMP-CMP kinase). This is a downstream consequence rather than a direct function. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Pyrimidine metabolism; UMP biosynthesis via de novo pathway |
| GO:0044205 'de novo' UMP biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: IEA annotation for de novo UMP biosynthesis. DHODH catalyzes step 4 of 6 in de novo UMP biosynthesis. This is an appropriate biological process annotation for the enzyme. Reason: Correct biological process. DHODH is specifically part of the de novo UMP biosynthetic pathway, providing orotate which is converted to OMP and then UMP by the bifunctional UMPS enzyme. UniProt pathway annotation confirms this. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt orotate from (S)-dihydroorotate (quinone route): step 1/1 |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: IDA annotation based on immunofluorescence data curation. Correct localization but less specific than GO:0005743 (mitochondrial inner membrane). The IDA evidence is valuable as experimental confirmation. Reason: Correct localization with experimental evidence. While less specific than inner membrane, IDA evidence from immunofluorescence appropriately resolves to mitochondrion level. This complements the more specific inner membrane annotations. Supporting Evidence: file:human/DHODH/DHODH-uniprot.txt Mitochondrion inner membrane |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: HTP annotation from the MitoCoP high-confidence mitochondrial proteome study. DHODH was identified in the MitoCoP proteome through multiple approaches including subtractive proteomics, spatial proteomics, and importomics. Reason: Correct localization with high-confidence proteomics evidence. PMID:34800366 used rigorous multi-method approach to define the mitochondrial proteome. DHODH is a bona fide mitochondrial protein confirmed by this study. Supporting Evidence: PMID:34800366 defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP) |
| GO:0106430 dihydroorotate dehydrogenase (quinone) activity | IDA PMID:8925840 Functional expression of a fragment of human dihydroorotate ... | ACCEPT | Summary: IDA annotation from the seminal characterization of recombinant human DHODH. This study purified the enzyme and demonstrated kinetics with ubiquinone substrates (Km 9.9 uM for ubiquinone-50), confirming the quinone-dependent mechanism. Reason: This is the definitive experimental demonstration of human DHODH catalytic activity with quinone electron acceptor. The kinetic parameters reported (Km 4 uM for dihydroorotate, Km 9.9 uM for ubiquinone-50) establish EC 1.3.5.2 classification. This is a core annotation with direct experimental evidence. Supporting Evidence: PMID:8925840 By kinetics analysis, Km values for dihydroorotate and ubiquinone-50 were found to be 4 microM and 9.9 microM, respectively, while Km values for dihydroorotate and decylubiquinone were 9.4 microM and 13.7 microM, respectively |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-73569 | ACCEPT | Summary: TAS annotation from Reactome pathway curation. Reactome reaction R-HSA-73569 represents "DHODH:FMN oxidises (S)-DHO to orotate" occurring at the inner mitochondrial membrane. Reason: Correct localization from expert pathway curation. Reactome has curated DHODH function in pyrimidine biosynthesis with appropriate cellular context. Supporting Evidence: Reactome:R-HSA-73569 DHODH:FMN oxidises (S)-DHO to orotate |
| GO:0005743 mitochondrial inner membrane | TAS Reactome:R-HSA-9708759 | ACCEPT | Summary: TAS annotation from Reactome for DHODH inhibitor binding. This Reactome entry covers pharmacological targeting of DHODH at the inner membrane. Reason: Correct localization. The inhibitor binding site is in the ubiquinone tunnel at the interface between the N-terminal membrane helices and the catalytic TIM barrel, consistent with inner membrane localization. Supporting Evidence: Reactome:R-HSA-9708759 DHODH:FMN binds DHODH inhibitors |
| GO:0110076 negative regulation of ferroptosis | IDA PMID:33981038 DHODH-mediated ferroptosis defence is a targetable vulnerabi... | NEW | Summary: DHODH functions as a parallel ferroptosis suppression pathway independent of GPX4 and FSP1. By reducing ubiquinone to ubiquinol (CoQH2) in the inner mitochondrial membrane, DHODH generates a radical-trapping antioxidant that prevents lipid peroxidation in mitochondrial membranes. This function was demonstrated by Mao et al. 2021 (Nature) showing that DHODH inhibition sensitizes cancer cells to ferroptosis. Reason: Well-established function not currently annotated. DHODH-mediated production of CoQH2 provides antioxidant protection against mitochondrial lipid peroxidation, complementing the cytosolic GPX4/FSP1 ferroptosis defense systems. This is mechanistically linked to DHODH's core enzyme activity. Proposed replacements: negative regulation of ferroptosis Supporting Evidence: PMID:33981038 Mechanistically, DHODH operates in parallel to mitochondrial GPX4 (but independently of cytosolic GPX4 or FSP1) to inhibit ferroptosis in the mitochondrial inner membrane by reducing ubiquinone to ubiquinol (a radical-trapping antioxidant with anti-ferroptosis activity) file:human/DHODH/DHODH-deep-research-falcon.md DHODH's respiratory-chain coupling via ubiquinone and membrane tunnel is well established and central to its physiology and pharmacology |
| GO:0010181 FMN binding | IDA PMID:10673429 Structures of human dihydroorotate dehydrogenase in complex ... | NEW | Summary: DHODH binds FMN (flavin mononucleotide) as its prosthetic group. Crystal structures (PDB: 1D3G) show FMN bound in the TIM barrel catalytic domain. UniProt records FMN binding based on PMID:10673429. Note that some annotations incorrectly list FAD binding - DHODH uses FMN, not FAD. Reason: FMN binding is experimentally demonstrated by X-ray crystallography and fluorimetric analysis. This is a core molecular function for DHODH - the FMN cofactor accepts electrons from dihydroorotate before transferring them to ubiquinone. Proposed replacements: FMN binding Supporting Evidence: PMID:8925840 Fluorimetric cofactor analysis revealed the presence of FMN in recombinant dihydroorotate dehydrogenase file:human/DHODH/DHODH-uniprot.txt Name=FMN; Xref=ChEBI:CHEBI:58210 |
| GO:0048038 quinone binding | IDA PMID:10673429 Structures of human dihydroorotate dehydrogenase in complex ... | NEW | Summary: DHODH contains a dedicated lipophilic ubiquinone-binding tunnel formed by its N-terminal membrane-associated helices. Crystal structures demonstrate that ubiquinone (coenzyme Q) and clinical inhibitors (brequinar, leflunomide active metabolite A77 1726) share a common binding site in this tunnel, directly coupling pyrimidine synthesis to the mitochondrial respiratory chain. Reason: The ubiquinone-binding tunnel is a structurally and functionally distinct feature of class-2 DHODHs (PMID:10673429; UniProt Q02127 COFACTOR/RHEA:30187). GO:0048038 (quinone binding) captures the electron-acceptor binding site that is separate from FMN binding (GO:0010181) and completes the mechanistic description of the catalytic cycle. This is a core molecular function linked to the catalytic activity GO:0106430. Proposed replacements: quinone binding Supporting Evidence: PMID:10673429 an alpha-helical domain that forms the opening of a tunnel leading to the active site. Both inhibitors share a common binding site in this tunnel file:human/DHODH/DHODH-uniprot.txt Reaction=(S)-dihydroorotate + a quinone = orotate + a quinol |
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Q: What is the physiological significance of DHODH protein-protein interactions?
Q: How does DHODH expression/activity correlate with ferroptosis sensitivity in different cell types?
Experiment: Structure-function studies of Miller syndrome variants to understand residual activity
Experiment: Systematic analysis of DHODH interactome to identify regulatory partners
Experiment: Metabolomics studies of DHODH inhibition effects on pyrimidine pools
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