DHODH

Summary: Specify the quinone-dependent activity within the valid PAINT inheritance.

Reason: Human recombinant DHODH directly oxidizes dihydroorotate with ubiquinone-50 or decylubiquinone as electron acceptor (PMID:8925840). The structural study resolves FMN, orotate and inhibitors, while the quinone pose is modeled (PMID:10673429). GO:0106430 specifies this quinone-dependent reaction within the broader inherited activity. The actual PTHR48109 tree places Q02127 beneath PTN000234184; its IBD is grounded in experimental descendant annotations, including the target. Target-derived seed evidence is legitimate, and there is no evidence of loss from this clade.

Proposed replacements: dihydroorotate dehydrogenase (quinone) activity

Summary: Retain the inherited mitochondrial inner-membrane location.

Reason: Human DHODH descends from PTN000234186, whose cellular-component assertion has experimental descendant grounding. The original import paper demonstrates human protein import into the yeast intermembrane space; endogenous rat protease/carbonate experiments establish membrane integration and sidedness in rat. Together with human mitochondrial proteomics and curated target-specific Reactome entities, these support the location without treating the rat deletion assays as direct human topology tests.

Summary: Retain de novo pyrimidine nucleobase biosynthesis.

Reason: DHODH performs the oxidation producing the pyrimidine base orotate. It therefore executes a chemical step of this process; it is not merely a substrate consumed by another enzyme. The PTN000234184 process assertion and the target placement are intact. Current experimentally grounded descendant annotations include Q08210, P0A7E1 and G5EB61.

Summary: Specify de novo UMP synthesis within pyrimidine ribonucleotide biosynthesis.

Reason: The broader ancestral process is biologically sound. Human DHODH catalyzes the dihydroorotate-to-orotate step of de novo UMP biosynthesis; it does not catalyze every pyrimidine ribonucleotide pathway. GO:0044205 provides the supported pathway scope and is a descendant of this broader process. This refinement does not reject the valid PAINT node or duplicate an annotation with a new row.

Proposed replacements: 'de novo' UMP biosynthetic process

Summary: Refine the automated broad enzyme activity to the quinone-specific reaction.

Reason: Human recombinant DHODH directly oxidizes dihydroorotate with ubiquinone-50 or decylubiquinone as electron acceptor (PMID:8925840). The structural study resolves FMN, orotate and inhibitors, while the quinone pose is modeled (PMID:10673429). GO:0106430 specifies this quinone-dependent reaction within the broader inherited activity. The inspected ARBA00047233 rule has a condition set requiring IPR001295, IPR005720 and IPR013785, all matched by Q02127. IPR005719 also maps the general dehydrogenase activity; the mapping is broad rather than a different paralog assignment.

Proposed replacements: dihydroorotate dehydrogenase (quinone) activity

Summary: Refine cytoplasm rather than reject it as incompatible with mitochondria.

Reason: GO:0005737 cytoplasm includes organelles and is not synonymous with cytosol. The verified typed path from mitochondrial inner membrane passes through mitochondrion to cytoplasm. Consequently the IPR005720 domain mapping is broad, not contradicted by mitochondrial localization. Refine to the independently supported inner membrane; do not infer that other experimentally detected pools are impossible.

Proposed replacements: mitochondrial inner membrane

Summary: Retain the UniProt subcellular-location mapping.

Reason: The source UniProt mitochondrial inner-membrane location agrees with target-specific Reactome DHODH:FMN complexes and independent mitochondrial evidence. Detailed N-terminal topology features are annotated by homology; the rat deletion experiments do not establish their boundaries directly in the current 395-residue human sequence.

Summary: Retain the InterPro-supported pyrimidine nucleobase biosynthetic role.

Reason: The target matches IPR005719 and IPR001295, whose actual GO mappings include this process. Oxidation of dihydroorotate directly creates orotate, so the target executes a step of nucleobase biosynthesis. Conserved domain evidence and direct human enzyme kinetics agree.

Summary: Specify de novo UMP biosynthesis within the broad keyword process.

Reason: The historical keyword-derived pyrimidine nucleotide biosynthesis annotation is compatible with the enzyme. Its specific contribution is oxidation of dihydroorotate during de novo UMP synthesis. GO:0044205 captures that contribution, rather than all pyrimidine nucleotide biosynthesis. The historical imported source identity is preserved; the separately dated GOA comparison is documented in the evidence guide.

Proposed replacements: 'de novo' UMP biosynthetic process

Summary: Specify the mitochondrial inner membrane.

Reason: The broad membrane annotation is correct for DHODH. IPR005719 maps this location class and Q02127 matches that family; original import/localization and curated human DHODH entities justify the more specific inner membrane. Existing specific rows do not make this broad source biologically excessive.

Proposed replacements: mitochondrial inner membrane

Summary: Specify the quinone-dependent oxidoreductase activity.

Reason: Human recombinant DHODH directly oxidizes dihydroorotate with ubiquinone-50 or decylubiquinone as electron acceptor (PMID:8925840). The structural study resolves FMN, orotate and inhibitors, while the quinone pose is modeled (PMID:10673429). GO:0106430 specifies this quinone-dependent reaction within the broader inherited activity. The UniProt oxidoreductase keyword is true but underspecified, and the historical imported source identity remains intact.

Proposed replacements: dihydroorotate dehydrogenase (quinone) activity

Summary: Specify dihydroorotate oxidation with quinone.

Reason: The IPR005720 and IPR001295 mappings correctly identify oxidation of a CH-CH group. Human DHODH supplies the more precise substrate and electron acceptor in GO:0106430. Broad family terminology is not evidence of a wrong target or paralog; the refinement is grounded in direct human chemistry.

Proposed replacements: dihydroorotate dehydrogenase (quinone) activity

Summary: Retain the specific quinone-dependent catalytic activity.

Reason: The combined automated mapping agrees with EC 1.3.5.2 and the current GO reaction. Recombinant human enzyme kinetics with ubiquinone-50 and decylubiquinone provide direct support. The current full-length product is 395 residues, whereas historical purification and structure studies used engineered fragments; these are not separate physiological isoforms.

Summary: Retain the verified DHODH–TTPA association as non-core.

Reason: The exact human pair has HuRI validated, array and pool Y2H records. Canonical sequence ROGIDs match 395-residue DHODH and 278-residue TTPA; main Methods describe quadruplicate pairwise retesting and sequence confirmation. TTPA is an alpha-tocopherol transfer protein. These records support association but do not establish a native DHODH vitamin-transfer mechanism, cellular compartment or stoichiometry, and no more specific supported partner-class binding term was established. Genericity alone does not justify removal or over-annotation under the explicit project instruction.

Summary: Refine RNF19B association to ubiquitin-protein ligase binding.

Reason: The exact human DHODH–RNF19B pair has validated, array and pool HuRI records with canonical sequence ROGIDs. The RNF19B record specifies residues 185–732 as sufficient binding region, so full native protein testing is not assumed. RNF19B is an experimentally supported E3 ubiquitin ligase: human RNF19B constructs show ubiquitination activity in PMID:27485036. GO:0031625 describes binding this partner class; it does not assign ubiquitination to DHODH, show that DHODH is a substrate, or establish physiological colocalization. The fragment and Y2H limits remain explicit.

Proposed replacements: ubiquitin protein ligase binding

Summary: Reject the electronic dihydroorotase assignment on reaction and target-family evidence.

Reason: GO:0004151 describes hydrolytic opening of dihydroorotate to carbamoyl-aspartate (EC 3.5.2.3), whereas human DHODH catalyses quinone-dependent oxidation to orotate. The target matches class-2 DHODH IPR005719 at residues 39–394 and its catalytic domain IPR005720 at 77–377; human enzyme kinetics and structures establish the FMN/quinone mechanism. This is incompatible with transferring the separate zinc-dependent dihydroorotase function, represented in human CAD by its DHOase-family machinery. The combined reaction, domain and structural evidence supports removal of this electronic inference without demanding experimental exclusion of every hypothetical secondary activity. The historical mouse donor record no longer has this term in the complete 2026-10-17 12:34:11 UTC snapshot, but the exact cause of the historical transfer remains untraced; no name-confusion or wrong-paralog claim is made.

Summary: Retain participation in the UDP-producing biosynthetic pathway.

Reason: The mouse O35435 GOA snapshot completed 2026-10-17 12:34:11 UTC contains curator-backed experimental UDP-biosynthesis assertions to PMID:30449682 and PMID:6249586. The full 30449682 study uses mouse 4T1/B16 knockout and mouse cDNA reconstitution and measures DHODH activity, orotate and isotope incorporation into UMP. Its tracer endpoint is UMP, not a DHODH-catalysed UMP-to-UDP kinase reaction. GO:0006225 covers reactions and pathways producing UDP; DHODH itself catalyses the upstream orotate-producing step of this de novo route, supported by conserved human enzyme chemistry. This is catalytic pathway participation, not merely a perturbation consequence. Retain ACCEPT and include the existing UDP process alongside de novo UMP biosynthesis in the same core. Neither process term is an ancestor of the other, so UMP is not a narrower descendant replacement for UDP.

Summary: Retain de novo UMP biosynthesis as the core pathway.

Reason: DHODH catalyzes dihydroorotate oxidation to orotate in de novo UMP biosynthesis. This is an executed chemical step, not a substrate-only or generic necessity relationship. The actual GO definition includes the dihydroorotate oxidation route, consistent with human kinetics, UniProt pathway information and the target-specific Reactome reaction.

Summary: Retain the HPA mitochondrial immunofluorescence location at its measured resolution.

Reason: The current official HPA record lists mitochondrial localization as enhanced and also lists nucleoplasm and cytosol. Antibody HPA010123 has mitochondrial calls in MCF7 and U2OS; HPA011942 has mitochondrial calls in A549 and MCF7 alongside other locations. These reported antibody/cell-line results support mitochondrion but do not resolve an inner-membrane subcompartment or an isoform. Individual image pixels were not re-evaluated. The additional reported pools are not dismissed because a mitochondrial catalytic role is established.

Summary: Retain mitochondrial localization from the actual MitoCoP target record.

Reason: Published Table S1(A) row 301 contains DHODH/Q02127, protein group 5570, with MitoCoP inclusion and positive subtractive, spatial and importomics classifications; Table S1(B) row 274 corroborates the target. The original Methods combine human HEK293 fractionation and HeLa TOMM40 perturbation, with additional carbonate extraction. This protein-group evidence supports mitochondrion and membrane association, not exact inner-membrane topology or a separate official isoform for every protein identifier in the group.

Summary: Retain direct human quinone-dependent enzyme activity.

Reason: The available primary abstract explicitly reports purification of a recombinant human DHODH fragment expressed with a baculovirus system, FMN analysis and kinetics with dihydroorotate plus ubiquinone-50 or decylubiquinone. This is direct positive evidence for the existing IDA claim. The full paper was not recovered, so fragment endpoints and unreported controls are not invented. Independent human structural evidence supports the same catalytic framework.

Summary: Retain the location of the actual Reactome DHODH catalyst.

Reason: Reaction R-HSA-73569 uses catalyst activity 73535 and complex R-HSA-73534. Its DHODH component R-HSA-73523 resolves to UniProt Q02127, residues 1–395, in the mitochondrial inner membrane. This is target-specific compartment evidence, distinct from the cytosolic metabolite compartments in the reaction.

Summary: Retain the target-specific inhibitor-binding complex location.

Reason: R-HSA-9708759 includes the same inner-membrane DHODH:FMN entity R-HSA-73534 used in the catalytic reaction. The entity resolves to human Q02127, so the compartment claim belongs to DHODH itself. Inhibitor-binding context does not supply an additional physiological core function, and the location annotation does not require assuming a quinone was directly observed in an inhibitor crystal.

Summary: Retain the historical ferroptosis proposal as unresolved and outside the core.

Reason: This row is a pre-existing NEW proposal, not an imported GOA annotation. The 2021 abstract and accessible extended-data captions report genetic knockout/reconstitution as well as pharmacological and CoQ-related experiments; genetic evidence is not absent. However, the full main study was not available. The original 2023 Matters Arising demonstrates FSP1 inhibition by high brequinar concentrations and a modest, context-dependent DHODH-deletion effect in the tested systems, challenging the breadth and drug specificity of the original interpretation. The reply is accessible only through introductory text and extended captions, so the dispute cannot be adjudicated completely. Preserve the proposal identity without presenting universal, GPX4/FSP1-independent ferroptosis defence as a settled core or categorically denying context-dependent protection. The former active proposal is explicitly retired while its biological claim remains unresolved; this does not claim that GOA ever carried or retired the annotation.

Summary: Retain the historical FMN-binding proposal as part of the catalytic mechanism.

Reason: This is one of the three inherited proposals; no additional assertion is created. The genuine original human DHODH structure contains FMN and orotate, corroborated by the positive cofactor analysis in PMID:8925840. The crystallized construct was reported historically as Met30–Arg396 with an N-terminal ten-histidine tag; that engineered numbering does not revise the official 395-residue human product. FMN binding belongs to the same integrated catalytic unit rather than a second independent core.

Summary: Retain the historical binding proposal as an active, specific ubiquinone-binding assertion.

Reason: This authored proposal is refined from its historical GO:0048038/IDA/PMID:10673429 form, recorded in the notes, to GO:0048039 with direct human substrate evidence. Purified recombinant human DHODH has measured kinetics with ubiquinone-50 and decylubiquinone (PMID:8925840), establishing engagement of this electron-acceptor substrate class. The paper remains abstract-only, but these positive human biochemical findings are explicit. PMID:10673429 provides complementary tunnel/active-site structural context: FMN, orotate and inhibitors are resolved, while quinone geometry is modeled rather than a bound quinone observed in the crystal. Ubiquinone binding is a component of the existing catalytic core. This active NEW proposal replaces the historical broad proposal in the same position; no redundant parent or additional binding row is added.