D2HGDH

Summary: The domain mapping correctly indicates catalysis, already represented by the specific dehydrogenase activity.

Reason: Current InterPro target matches cover the C-terminal FAD-linked oxidase domain and superfamily. They support the original broad mapping; independent human assays establish GO:0051990, already accepted in this review, which entails catalytic activity. This action concerns informativeness, not a failed mapping or negative catalytic evidence.

Summary: Human mitochondrial proteomics directly supports the broad location.

Reason: The actual PMID:34800366 Table S1 protein group 8117, sheet A row 290, contains Q8N465 and class 1 subtractive proteomics, cluster 1 spatial profiling and class 1 importomics; sheet B row 263 includes D2HGDH in MitoCoP. The same row classifies it as peripheral, but its matrix annotation column is imported database context, not a new matrix-localization measurement. The source remains positive for mitochondria. The existing accepted mitochondrial-matrix annotation entails this broader compartment. The broad evidence is retained without narrowing its own experimental resolution. The human Peripheral category describes extraction-dependent membrane association, not a mutually exclusive submitochondrial compartment. It does not contradict matrix residence or establish permanent integral-membrane insertion. The separate rat freeze–thaw observation in PMID:15070399 supports readily releasable enzyme and an inferred soluble matrix pool; the species and extraction protocols are not equated.

Summary: The mitochondrial PAINT inheritance is supported by the actual target lineage.

Reason: The actual PTHR43716 tree places the exact 521-residue human target PTN002536236 below PTN000202300. The literal mitochondrial IBD dated 2017-02-28 has the same rat, yeast and human donor set; current complete donor records contain experimental mitochondrial annotations, and no negative target-path record overturns it. The human self donor is legitimate descendant evidence. The preserved is_active_in relation is not rewritten into a direct human fractionation result. The existing accepted mitochondrial-matrix annotation entails this broader compartment. The broad evidence is retained without narrowing its own experimental resolution.

Summary: The Human Protein Atlas supports mitochondrial staining.

Reason: The current HPA record reports supported mitochondrial ICC/IF localization, with antibody HPA060548 and mitochondrial locations for Hep-G2 and RT-4 cells. This is gene-level imaging evidence; neither matrix resolution nor product-specific antibody recognition was established here. The existing accepted mitochondrial-matrix annotation entails this broader compartment. The broad evidence is retained without narrowing its own experimental resolution.

Summary: The UniProt location mapping is consistent with independent localization evidence.

Reason: The literal SL-0173 mapping names mitochondria. Its biological support comes from rat fractionation, human MitoCoP proteomics and curated HPA ICC/IF, rather than treating a UniProt location label as an independent experiment. The existing accepted mitochondrial-matrix annotation entails this broader compartment. The broad evidence is retained without narrowing its own experimental resolution.

Summary: The rat mitochondrial fractionation supports the human orthology-based location inference.

Reason: The P84850 donor is rat D2hgdh. PMID:15070399 detects activity in rat liver mitochondrial fractions, while the human targeting sequence in that paper is a prediction. This source supports a mitochondrial ISS assertion; it does not itself establish a human matrix subcompartment or a measured precursor-cleavage site. The existing accepted mitochondrial-matrix annotation entails this broader compartment. The broad evidence is retained without narrowing its own experimental resolution.

Summary: Reactome locates the D2HGDH catalytic entity in the mitochondrial matrix.

Reason: The source reaction R-HSA-880007 contains catalyst 880022, whose physical entity 880006 is human Q8N465 in GO:0005759. Independent PMID:15070399 rat liver fractionation places activity in mitochondrial fractions, and Discussion reports ready release by freezing–thawing, from which the authors infer probable soluble matrix residence. This supports the human curated matrix location by orthologous biochemical evidence, not a direct human matrix assay. Human MitoCoP Table S1 classifies D2HGDH as Peripheral using carbonate/sonication extraction, which addresses membrane association and need not exclude a matrix-facing or readily releasable pool. Neither source resolves the fraction freely soluble in living human cells. Reactome models residues 14–521 from a predicted transit-peptide boundary; that cleavage site is not experimentally established here.

Summary: D-lactate oxidation is a measured secondary substrate activity.

Reason: The purified rat enzyme oxidizes D-lactate in the PMS/DCIP assay, and recombinant human residues 51–521 also show weak but positive D-lactate turnover. The current lactate-metabolism definition includes chemical reactions involving lactate and does not restrict the term to L-lactate. Retain the performed reaction as non-core; neither weak turnover nor stereochemical difference is evidence of absence. The experiments do not establish an important physiological D-lactate clearance role. This is not a numerical cutoff based only on comparison with the negligible L-2-hydroxyglutarate signal: the human study reports D-lactate-dependent kinetics and measurable substrate binding, corroborated by the earlier rat turnover and competition experiments. The human specific activity is low (0.16 versus 2.02 micromol/min/mg for D-2-hydroxyglutarate), and the reported kcat is 13.5-fold lower. These support a weak performed reaction, not an important physiological clearance pathway; no new L-enantiomer function is inferred.

Summary: D2HGDH performs the oxidation that produces 2-oxoglutarate.

Reason: The actual Reactome pathway R-HSA-880009 contains reaction 880007 and the Q8N465 catalyst path. D2HGDH itself oxidizes D-2-hydroxyglutarate to 2-oxoglutarate, so it participates directly in this metabolic process. Other pathway enzymes and opposite-enantiomer reactions are not transferred to D2HGDH. The model uses FAD/FADH2 to describe cofactor redox chemistry, not a demonstrated physiological electron-acceptor protein.

Summary: Human D2HGDH directly oxidizes D-malate, with strong activity under the measured conditions.

Reason: PMID:33431826 directly assays purified human residues 51–521 with ZnCl2, PMS and DCIP at 37 degrees C. D-malate turnover is comparable to, and slightly exceeds, D-2-hydroxyglutarate turnover at the stated substrate concentration; the structure also contains D-malate in the substrate pocket. Retain the malate process as a supported non-core substrate reaction because its physiological flux is not established, not because its in-vitro activity is weak. Do not convert this D-malate result into L-malate/TCA-cycle activity or an NAD-dependent decarboxylating reaction.

Summary: The human enzyme coordinates zinc at its catalytic substrate-binding site.

Reason: The ligand-bound crystal structures of human residues 51–521 contain a metal modeled as Zn2+ after ZnCl2 soaking, with defined coordination by His434, His441, Glu475, FAD and substrate. Mutagenesis supports the catalytic importance of these residues. This is direct support for zinc binding; a unique in-vivo metallation state or a zinc-response pathway is not established by the structures.

Summary: The positive manganese effect is enzyme modulation, while the broad cellular response claim exceeds this experiment.

Reason: PMID:15070399 directly reports increased activity of purified rat enzyme when bivalent ions are added to the radiochemical assay. This positive biochemical result must be preserved. The current GO response definition, however, concerns a change in the state or activity of a cell or organism after an ion stimulus; no corresponding intact-cell or organism response process is demonstrated here. Mark the ISS process inference as over-annotated without calling the metal effect false or claiming that D2HGDH cannot participate in such a response. Zinc binding is independently captured by the existing molecular-function annotation.

Summary: The positive zinc effect is enzyme modulation, while the broad cellular response claim exceeds this experiment.

Reason: PMID:15070399 directly reports increased activity of purified rat enzyme when bivalent ions are added to the radiochemical assay. Human full-ORF transfected-cell lysates also show zinc and cobalt activation. This positive biochemical result must be preserved. The current GO response definition, however, concerns a change in the state or activity of a cell or organism after an ion stimulus; no corresponding intact-cell or organism response process is demonstrated here. Mark the ISS process inference as over-annotated without calling the metal effect false or claiming that D2HGDH cannot participate in such a response. Zinc binding is independently captured by the existing molecular-function annotation.

Summary: The native protein is a degradation target, and its N terminus contributes susceptibility in engineered constructs.

Reason: PMID:33053349 goes beyond a proteomic hit: VbP depletes native/full-length D2HGDH; proteasome and cullin-pathway inhibitors block depletion; full human 1–521 is more protease-sensitive than 52–521, and deletion of the N terminus prevents VbP-induced degradation. Transferring residues 1–51 onto CARD8-ZUC permits DPP8/9-inhibitor-triggered GSDMD cleavage and LDH release in CARD8-null THP-1 cells. These positive deletion and fusion results establish a transferable susceptibility element, not a native D2HGDH role performing a protein-destabilization step. The broad gene-level process attribution is therefore over-annotated; this does not deny the engineered cis effect or infer absence of degradation.

Summary: The positive cobalt effect is enzyme modulation, while the broad cellular response claim exceeds this experiment.

Reason: PMID:15070399 directly reports increased activity of purified rat enzyme when bivalent ions are added to the radiochemical assay. Human full-ORF transfected-cell lysates also show zinc and cobalt activation. This positive biochemical result must be preserved. The current GO response definition, however, concerns a change in the state or activity of a cell or organism after an ion stimulus; no corresponding intact-cell or organism response process is demonstrated here. Mark the ISS process inference as over-annotated without calling the metal effect false or claiming that D2HGDH cannot participate in such a response. Zinc binding is independently captured by the existing molecular-function annotation.

Summary: The negative magnesium enzyme result is established, but the broader human process exclusion is unresolved.

Reason: This original ISS assertion is explicitly NOT and retains the rat P84850 donor. PMID:15070399 reports no magnesium-dependent stimulation of purified rat enzyme in the tested radiochemical assay; that negative biochemical observation is accepted. The current GO definition concerns a cell or organism response, so the experiment neither establishes nor contradicts absence of human D2HGDH participation in that broader process. The deliberate asymmetry is epistemic: the three positive ion-response rows promote observed enzyme stimulation into an unshown cellular process and are over-annotated; these NOT rows instead require adjudicating a broader exclusion, whose truth remains unresolved. UNDECIDED concerns that cross-species process exclusion, not access to the fully read paper or doubt about the negative assay. It does not endorse a universal absence, imply a positive response, or require removal of the experimental observation.

Summary: FAD is the tightly bound redox cofactor of human D2HGDH.

Reason: Human residues 51–521 co-purify with FAD, and every reported crystal structure contains noncovalently bound FAD in the catalytic pocket. The protein-side contacts and catalytic mechanism support this cofactor-binding activity. GO:0050660 covers oxidized or reduced FAD, whereas GO:0071949 specifically names the oxidized form; they are not duplicate identifiers. Preserve both existing assertions but do not create a separate FAD-binding core unit. The current InterPro records contain the literal corresponding GO mappings and exact Q8N465 domain matches; their classification is corroborated by the human structures. This retention is a source-level cofactor judgment rather than a claim that an ancestor adds independent logical coverage: the broader mapping describes the redox cofactor across the catalytic cycle, while the narrower mapping specifies oxidized FAD. TOO_GENERAL is used for uninformative generic catalysis and broad locations here, not as an automatic deletion of every supported ancestor. Both cofactor observations remain integrated into the single dehydrogenase core.

Summary: The negative calcium enzyme result is established, but the broader human process exclusion is unresolved.

Reason: This original ISS assertion is explicitly NOT and retains the rat P84850 donor. PMID:15070399 reports no calcium-dependent stimulation of purified rat enzyme in the tested radiochemical assay; that negative biochemical observation is accepted. The current GO definition concerns a cell or organism response, so the experiment neither establishes nor contradicts absence of human D2HGDH participation in that broader process. The deliberate asymmetry is epistemic: the three positive ion-response rows promote observed enzyme stimulation into an unshown cellular process and are over-annotated; these NOT rows instead require adjudicating a broader exclusion, whose truth remains unresolved. UNDECIDED concerns that cross-species process exclusion, not access to the fully read paper or doubt about the negative assay. It does not endorse a universal absence, imply a positive response, or require removal of the experimental observation.

Summary: This paper directly establishes human D-2-hydroxyglutarate dehydrogenase activity as well as rat enzyme chemistry.

Reason: PMID:15070399 purifies the rat enzyme, identifies 2-oxoglutarate as product, and then expresses the human full open reading frame in HEK293 cells. Human overexpression increases detritiation of labeled D/L-2-hydroxyglutarate, with D-enantiomer competition and zinc/cobalt activation supporting the identity. The source is not misattributed merely because most purified-enzyme characterization is in rat. Artificial electron acceptors and slower oxygen-dependent turnover do not establish the physiological electron-acceptor partner.

Summary: Wild-type and disease-mutant human enzyme assays directly support the reaction.

Reason: The cached abstract reports reduced D-2-hydroxyglutarate dehydrogenase activity after expression of human missense-mutant proteins in HEK-293 cells. This direct functional result supports the EXP annotation independently of the clinical association. The separately recovered original PMC1196381 Table 1 compares wild-type enzyme with Val444Ala and Ile147Ser. Its assay details and exact public original locator are recorded in D2HGDH-primary-source-notes.md; that authored note is navigation to the original, not independent experimental evidence. The cache remains abstract-only and unchanged, while full_text_unavailable remains false because the original full text was actually recovered and read.

Summary: Human disease-mutant expression supports the established dehydrogenase activity.

Reason: The complete PMID:16037974 abstract reports markedly reduced enzyme activity after mutant overexpression, including the N439D variant, in a study of mild D-2-hydroxyglutaric aciduria. The full experimental protocol was not independently recovered. Retain the curator-assigned EXP for a function independently established by human expression and structural biochemistry; do not invent assay conditions or infer that every clinical phenotype is a directly performed D2HGDH process.

Summary: This paper directly establishes human D-2-hydroxyglutarate dehydrogenase activity as well as rat enzyme chemistry.

Reason: PMID:15070399 purifies the rat enzyme, identifies 2-oxoglutarate as product, and then expresses the human full open reading frame in HEK293 cells. Human overexpression increases detritiation of labeled D/L-2-hydroxyglutarate, with D-enantiomer competition and zinc/cobalt activation supporting the identity. The source is not misattributed merely because most purified-enzyme characterization is in rat. Artificial electron acceptors and slower oxygen-dependent turnover do not establish the physiological electron-acceptor partner.

Summary: The combined electronic mapping agrees with independently measured enzyme chemistry.

Reason: Current ARBA00089517 branch 0 requires three FunFam matches, all present in the official Q8N465 record; the Saccharomycotina-specific alternative branch does not match human. The literal RHEA:38295 equation is D-2-hydroxyglutarate plus an unspecified acceptor yielding 2-oxoglutarate and reduced acceptor. These source premises are explicitly reconstructed, and independent human assays support the reaction. EC, Rhea and UniProt reaction labels are not treated as new experimental proof.

Summary: Patient enzyme activity measurements support the catalytic function.

Reason: The complete PMID:20020533 abstract reports D2HGDH mutations in 24 of 50 patients. All mutation-positive patients had impaired enzyme activity, and patients with normal activity had no detected mutations. This does not establish 24 distinct mutations, mutation-specific expression assays, or normal activity in every mutation-negative patient. The detailed protocol was not independently recovered; these patient measurements and independent human chemistry support retaining this curator-assigned IMP without adding biological processes from clinical association.

Summary: Active-site mutagenesis establishes the human D-2-hydroxyglutarate catalytic machinery.

Reason: In the recombinant human 51–521 construct, mutations of zinc/substrate-coordinating residues abolish or severely impair D-2-hydroxyglutarate oxidation. The paper reports circular-dichroism controls for these selected active-site mutants, supporting a catalytic interpretation rather than automatically attributing every effect to misfolding. Disease-variant behavior is heterogeneous and should not be generalized from this subset. The C-terminal substrate and metal sites belong to the tested long product; the two shorter official splice products were not assayed.

Summary: FAD is the tightly bound redox cofactor of human D2HGDH.

Reason: Human residues 51–521 co-purify with FAD, and every reported crystal structure contains noncovalently bound FAD in the catalytic pocket. The protein-side contacts and catalytic mechanism support this cofactor-binding activity. GO:0050660 covers oxidized or reduced FAD, whereas GO:0071949 specifically names the oxidized form; they are not duplicate identifiers. Preserve both existing assertions but do not create a separate FAD-binding core unit. The current InterPro records contain the literal corresponding GO mappings and exact Q8N465 domain matches; their classification is corroborated by the human structures. This retention is a source-level cofactor judgment rather than a claim that an ancestor adds independent logical coverage: the broader mapping describes the redox cofactor across the catalytic cycle, while the narrower mapping specifies oxidized FAD. TOO_GENERAL is used for uninformative generic catalysis and broad locations here, not as an automatic deletion of every supported ancestor. Both cofactor observations remain integrated into the single dehydrogenase core.

Summary: Meso-tartrate oxidation is a supported secondary activity inferred from the rat ortholog.

Reason: Purified rat D2hgdh oxidizes meso-tartrate in the PMS/DCIP assay, and meso-tartrate competes in the labeled D-2-hydroxyglutarate assay. The original reports the turnover as results not shown, which is still a positive reported experiment; D- and L-tartrate were poor inhibitors. Retain the ISS broad tartrate-metabolism assertion as non-core, with explicit meso-isomer and donor scope. The inspected human structural study does not assay tartrate, and no physiological tartrate-disposal role is established.