{"gene":"CAT","run_date":"2026-06-14T21:01:35+00:00","timeline":{"discoveries":[{"year":1979,"finding":"Two distinct acyl-CoA:glycine N-acyltransferases were purified to near-homogeneity from bovine liver mitochondria, each consisting of a single polypeptide chain (~33 kDa). Preliminary kinetic studies were consistent with a sequential (ordered) reaction mechanism in which acyl-CoA binds first, glycine adds before CoA leaves, and the peptide product dissociates last.","method":"Enzyme purification, substrate specificity assays, stoichiometric cleavage assays, mass spectrometry of products, kinetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — near-homogeneity purification, stoichiometric product assays, kinetic mechanism established; replicated across two enzymes in same study","pmids":["457678"],"is_preprint":false},{"year":1977,"finding":"Acyl-CoA:glycine N-acyltransferase from beef liver is composed of a single polypeptide (~35 kDa) with one active site per molecule, as demonstrated by photoaffinity labeling with p-azidobenzoyl-CoA. Benzoyl-CoA protects the enzyme from photoinactivation, confirming that p-azidobenzoyl-CoA targets the acyl-CoA binding active site.","method":"Photoaffinity labeling, UV irradiation, competitive protection assay with benzoyl-CoA","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site-directed photoaffinity labeling with competitive protection; single lab but multiple orthogonal methods (alternate substrate activity, photolabeling, protection)","pmids":["889778"],"is_preprint":false},{"year":1986,"finding":"Acyl-CoA:glycine N-acyltransferase is exclusively an intramitochondrial enzyme in rat liver; no activity was detected in peroxisomes despite their capacity to produce acyl-CoAs.","method":"Subcellular fractionation, organelle-specific enzyme activity assays","journal":"Biochemical medicine and metabolic biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation with activity measurement; single lab but direct localization experiment","pmids":["3741707"],"is_preprint":false},{"year":1994,"finding":"Human mitochondrial acyl-CoA:glycine N-acyltransferase (ACGNAT) was purified to homogeneity from adult liver; it is a monomer of 30 kDa (pI 6.8). Km values for benzoyl-CoA, salicyl-CoA, isovaleryl-CoA, and octanoyl-CoA were determined, establishing the enzyme's substrate preference profile.","method":"Protein purification to homogeneity, SDS-PAGE, isoelectric focusing, enzyme kinetics","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — purification to homogeneity with biochemical characterization; single lab, multiple orthogonal methods","pmids":["7802672"],"is_preprint":false},{"year":1996,"finding":"N-fatty acylglycines are enzymatically produced from fatty acyl-CoA and glycine by acyl-CoA:glycine N-acyltransferase, and N-myristoylglycine is a substrate for peptidylglycine alpha-amidating enzyme, establishing a two-enzyme biosynthetic route to fatty acid primary amides.","method":"In vitro enzyme assay with N-myristoylglycine substrate, measurement of (V/K)app","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro enzyme assay establishing substrate activity; single lab, single method for the GLYAT step","pmids":["8660675"],"is_preprint":false},{"year":2011,"finding":"The catalytic glutamate residue (Glu226 in bovine GLYAT) was identified as essential for activity: an E226Q mutant showed decreased activity at pH 8.0 that could be compensated by increasing pH, indicating Glu226 deprotonates glycine to facilitate nucleophilic attack on the acyl-CoA.","method":"Recombinant expression, E226Q active-site mutagenesis, enzyme kinetics at varying pH, protein modeling","journal":"Drug metabolism and disposition","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis combined with pH-rescue kinetics and molecular modeling; single lab but multiple orthogonal methods supporting the catalytic mechanism","pmids":["22071172"],"is_preprint":false},{"year":2012,"finding":"Single nucleotide polymorphisms in human GLYAT alter enzyme activity: N156S variant is more active, F168L is less active, and R199C is completely inactive. An E227Q active-site mutant is also inactive, confirming Glu227 as the catalytic residue in human GLYAT. The R199C inactivity is attributed to destabilization of an α-loop-α motif important for substrate binding in the GNAT superfamily.","method":"Site-directed mutagenesis of recombinant human GLYAT, expression, purification, enzymatic characterization, molecular modeling with CoA","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis (E227Q) confirming catalytic residue, SNP variant functional characterization, structural modeling; single lab, multiple orthogonal methods","pmids":["23237781"],"is_preprint":false},{"year":2012,"finding":"Human GLYAT is expressed specifically in liver and kidney, and the protein localizes to mitochondria, as confirmed by immunolabeling.","method":"Tissue-specific expression analysis, cellular immunolocalization with affinity-purified anti-GLYAT antibody","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct immunolocalization experiment with affinity-purified antibody; single lab, consistent with prior biochemical fractionation data","pmids":["22475485"],"is_preprint":false},{"year":2017,"finding":"Human GLYAT exhibits mechanistic kinetic cooperativity (sigmoidal substrate activation with glycine variable and substrate inhibition with benzoyl-CoA variable) consistent with a Ferdinand enzyme mechanism, rather than the previously assumed Michaelis-Menten or ping-pong mechanism.","method":"Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods, allosteric sigmoidal enzyme kinetic modeling","journal":"Journal of biochemical and molecular toxicology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — two orthogonal analytical methods; single lab; contradicts prior mechanistic models","pmids":["28759163"],"is_preprint":false},{"year":2021,"finding":"Human GLYAT wild-type protein localizes to the mitochondrial matrix when expressed as an eGFP fusion in HEK293 cells, confirming its intramitochondrial localization in a human cellular context. The p.(Gln61Leu) variant shows decreased specific activity and the p.(Asn156Ser) variant shows somewhat increased activity compared to wild-type.","method":"Recombinant expression in E. coli and HEK293 cells, eGFP fusion localization imaging, enzyme activity assay after purification","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live-cell fluorescence imaging for localization plus enzymatic activity characterization; single lab, two orthogonal approaches","pmids":["33567294"],"is_preprint":false},{"year":2021,"finding":"GLYAT exhibits cooperative substrate binding (two-substrate Hill kinetics) and the rare 156Asn>Ser,199Arg>Cys haplotype reduces kcat to ~9.8% of the most abundant haplotype while increasing affinity for benzoyl-CoA, demonstrating that Arg199 is critical for catalytic turnover.","method":"Recombinant expression of haplotype variants, kinetic characterization fitted to two-substrate Hill equation","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme kinetics with multiple haplotype variants; single lab, consistent with prior mutagenesis data on R199C","pmids":["33803916"],"is_preprint":false},{"year":2025,"finding":"The first crystal structure of bovine GLYAT was solved in apo form and bound to benzoyl-CoA. Structural analysis and mutagenesis of key residues revealed that catalysis proceeds via a general base mechanism driven by a potential low-barrier hydrogen bond (LBHB) formed between a catalytic Glu-His dyad, with Glu deprotonating the glycine amine for nucleophilic attack on the acyl-CoA thioester.","method":"X-ray crystallography (apo and benzoyl-CoA-bound structures), active-site mutagenesis, functional validation","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determination with bound substrate combined with mutagenesis of catalytic residues; multiple orthogonal methods in single rigorous study","pmids":["40938199"],"is_preprint":false},{"year":2025,"finding":"GLYAT is upregulated in dorsal root ganglion (DRG) neurons in a mouse osteoarthritis pain model. GLYAT overexpression increases reactive oxygen species (ROS) levels in DRG neurons and upregulates TRPV1, causing mechanical and thermal hyperalgesia; GLYAT loss-of-function alleviates pain-like behaviors. ROS clearance or TRPV1 blockade in GLYAT-overexpressing mice rescues hyperalgesia, placing GLYAT upstream of ROS→TRPV1 in pain signaling.","method":"Transcriptomic sequencing of DRG, viral overexpression and genetic knockout in mice, ROS measurement, TRPV1 immunolabeling, pharmacological rescue with ROS scavenger and TRPV1 blocker, behavioral nociception assays","journal":"The journal of pain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic gain- and loss-of-function with pharmacological rescue establishing pathway position; single lab, multiple orthogonal approaches","pmids":["40288509"],"is_preprint":false},{"year":2025,"finding":"A homozygous nonsense variant (p.Q108Ter) in human GLYAT abolishes GLYAT enzymatic activity in vitro, causing glycine N-acyltransferase deficiency associated with developmental delays, confirming that GLYAT enzymatic activity is required for normal glycine conjugation metabolism in humans.","method":"Whole exome sequencing, Sanger confirmation, in vitro activity assay of the nonsense variant","journal":"JIMD reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single patient, in vitro activity abolishment confirmed; establishes loss-of-function consequence but single case","pmids":["40747359"],"is_preprint":false}],"current_model":"Glycine N-acyltransferase (GLYAT) is a mitochondrial matrix enzyme (monomeric, ~30–35 kDa) that catalyzes the transfer of acyl groups from acyl-CoA donors (preferentially benzoyl-CoA and short/medium-chain acyl-CoAs) to glycine via a general base mechanism in which a catalytic Glu-His dyad (with a potential low-barrier hydrogen bond) deprotonates glycine to enable nucleophilic attack on the acyl-CoA thioester, releasing CoA and an N-acylglycine product; the enzyme displays cooperative (Ferdinand) kinetics, harbors a single active site per polypeptide, and its activity is modulated by key residues (Glu226/227, Arg199) whose mutation abolishes or severely reduces catalysis, while loss of GLYAT activity in vivo disrupts CoA homeostasis, glycine metabolism, and—in DRG neurons—promotes ROS accumulation and TRPV1 upregulation to drive pain sensitization."},"narrative":{"mechanistic_narrative":"GLYAT (acyl-CoA:glycine N-acyltransferase) is a mitochondrial matrix enzyme of liver and kidney that conjugates glycine to acyl-CoA donors, generating N-acylglycine products as a route for detoxification of benzoate and short/medium-chain acyl groups [PMID:7802672, PMID:22475485, PMID:33567294]. The purified enzyme is a single-chain monomer of ~30–35 kDa bearing one active site per polypeptide, with a preference for benzoyl-CoA and additional activity toward salicyl-, isovaleryl-, octanoyl-, and fatty acyl-CoA substrates [PMID:457678, PMID:889778, PMID:7802672]; by acting on fatty acyl-CoA it feeds a two-enzyme route to fatty acid primary amides via downstream peptidylglycine alpha-amidating enzyme [PMID:8660675]. Catalysis proceeds through a general base mechanism in which a catalytic glutamate (Glu226/Glu227) deprotonates the glycine amine to enable nucleophilic attack on the acyl-CoA thioester, a mechanism supported by pH-rescue of an E226Q/E227Q mutant and by the crystal structure of the enzyme bound to benzoyl-CoA revealing a Glu-His catalytic dyad [PMID:22071172, PMID:23237781, PMID:40938199]; Arg199 is essential for catalytic turnover, as its loss (R199C) abolishes activity by destabilizing a substrate-binding motif [PMID:23237781, PMID:33803916]. Rather than simple Michaelis-Menten or ping-pong behavior, GLYAT displays cooperative (Ferdinand-type) kinetics with sigmoidal glycine activation and benzoyl-CoA substrate inhibition [PMID:28759163, PMID:33803916]. Loss-of-function consequences are established in vivo: a homozygous nonsense variant (p.Q108Ter) abolishes activity and causes glycine N-acyltransferase deficiency with developmental delays in humans [PMID:40747359], and GLYAT upregulation in dorsal root ganglion neurons drives a ROS→TRPV1 axis that produces pain hypersensitivity in a mouse osteoarthritis model [PMID:40288509].","teleology":[{"year":1979,"claim":"Established that glycine conjugation is carried out by discrete acyl-CoA:glycine N-acyltransferase enzymes with a defined ordered kinetic mechanism, settling the basic identity and reaction logic of the activity.","evidence":"Purification to near-homogeneity from bovine liver mitochondria with kinetic mechanism analysis","pmids":["457678"],"confidence":"High","gaps":["Kinetic model later revised to cooperative rather than simple ordered","No structural basis for catalysis defined","Human enzyme not yet characterized"]},{"year":1977,"claim":"Demonstrated the enzyme is a single polypeptide with one acyl-CoA binding active site, defining its architecture and the location of the catalytic site.","evidence":"Photoaffinity labeling with p-azidobenzoyl-CoA plus competitive protection by benzoyl-CoA in beef liver enzyme","pmids":["889778"],"confidence":"High","gaps":["No residue-level identification of catalytic groups","No structure"]},{"year":1986,"claim":"Resolved the subcellular compartment of the activity by showing it is exclusively intramitochondrial, distinguishing it from peroxisomal acyl-CoA metabolism.","evidence":"Subcellular fractionation with organelle-specific activity assays in rat liver","pmids":["3741707"],"confidence":"Medium","gaps":["Submitochondrial location not pinpointed","Human localization not yet shown"]},{"year":1994,"claim":"Characterized the human liver enzyme and its substrate preference profile, anchoring later human genetic and mechanistic work to a defined monomeric protein.","evidence":"Purification to homogeneity and kinetic determination of Km for benzoyl-, salicyl-, isovaleryl-, and octanoyl-CoA from human liver","pmids":["7802672"],"confidence":"High","gaps":["Catalytic residues not identified","Gene/sequence-level variant effects unaddressed"]},{"year":1996,"claim":"Extended the enzyme's biological scope by showing it produces N-fatty acylglycines that feed a two-enzyme pathway to fatty acid primary amides.","evidence":"In vitro enzyme assay with N-myristoylglycine and downstream peptidylglycine alpha-amidating enzyme","pmids":["8660675"],"confidence":"Medium","gaps":["Physiological flux through this route not quantified","Single method for the GLYAT step"]},{"year":2012,"claim":"Identified the catalytic glutamate (Glu226/Glu227) and showed it acts as a general base, and demonstrated that natural human SNPs including the inactivating R199C alter activity, linking genotype to enzyme function.","evidence":"Active-site mutagenesis with pH-rescue kinetics, SNP variant characterization, and molecular modeling of recombinant bovine and human GLYAT","pmids":["22071172","23237781"],"confidence":"High","gaps":["Catalytic partner (His) of the dyad not yet defined","No experimental structure"]},{"year":2012,"claim":"Confirmed tissue-restricted expression (liver, kidney) and mitochondrial localization of the human protein by direct immunolabeling.","evidence":"Tissue expression analysis and immunolocalization with affinity-purified anti-GLYAT antibody","pmids":["22475485"],"confidence":"Medium","gaps":["Submitochondrial compartment not resolved by this method"]},{"year":2017,"claim":"Revised the kinetic model, showing GLYAT follows a cooperative Ferdinand mechanism rather than Michaelis-Menten or ping-pong, reframing how its activity is regulated by substrate.","evidence":"Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods with allosteric modeling","pmids":["28759163"],"confidence":"Medium","gaps":["Structural basis of cooperativity unknown","Single lab"]},{"year":2021,"claim":"Confirmed mitochondrial matrix localization in a human cellular context and quantified variant/haplotype effects, establishing Arg199 as critical for catalytic turnover.","evidence":"eGFP-fusion live-cell imaging in HEK293 and kinetic characterization of haplotype variants fitted to two-substrate Hill equations","pmids":["33567294","33803916"],"confidence":"Medium","gaps":["Mechanism by which R199 supports turnover inferred from modeling, not structure","Single lab"]},{"year":2025,"claim":"Provided the first atomic structure and definitive mechanism by capturing the benzoyl-CoA-bound enzyme and showing catalysis via a Glu-His dyad with a potential low-barrier hydrogen bond.","evidence":"X-ray crystallography of apo and benzoyl-CoA-bound bovine GLYAT with active-site mutagenesis","pmids":["40938199"],"confidence":"High","gaps":["Structure of human enzyme not solved","Structural basis of cooperative kinetics not directly addressed"]},{"year":2025,"claim":"Established physiological loss- and gain-of-function consequences: a nonsense variant causes human GLYAT deficiency with developmental delays, and GLYAT upregulation drives ROS→TRPV1 pain signaling in DRG neurons.","evidence":"Human exome sequencing with in vitro activity assay; mouse DRG overexpression/knockout with ROS and TRPV1 readouts and pharmacological rescue","pmids":["40747359","40288509"],"confidence":"Medium","gaps":["Single-patient disease evidence","Molecular link between GLYAT enzymatic output and ROS generation not defined","Pain phenotype shown in mouse only"]},{"year":null,"claim":"How GLYAT enzymatic activity mechanistically generates ROS and engages TRPV1, and how its cooperative kinetics are structurally encoded, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No biochemical link between acyl-CoA/glycine conjugation flux and ROS production","No structure rationalizing Ferdinand cooperativity","Human enzyme structure unsolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,3,5,6,11]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2,7,9]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,13]}],"complexes":[],"partners":[],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6IB77","full_name":"Glycine N-acyltransferase","aliases":["Acyl-CoA:glycine N-acyltransferase","AAc","Aralkyl acyl-CoA N-acyltransferase","Aralkyl acyl-CoA:amino acid N-acyltransferase","Benzoyl-coenzyme A:glycine N-acyltransferase","Glycine N-benzoyltransferase","HRP-1(CLP)"],"length_aa":296,"mass_kda":33.9,"function":"Mitochondrial acyltransferase which transfers an acyl group to the N-terminus of glycine and glutamine, although much less efficiently. Can conjugate numerous substrates to form a variety of N-acylglycines, with a preference for benzoyl-CoA over phenylacetyl-CoA as acyl donors. Thereby detoxify xenobiotics, such as benzoic acid or salicylic acid, and endogenous organic acids, such as isovaleric acid","subcellular_location":"Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q6IB77/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CAT","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CAT","total_profiled":1310},"omim":[{"mim_id":"621297","title":"LYMPHATIC ENDOTHELIAL TRANSCRIPTIONAL REGULATOR lncRNA 1; LETR1","url":"https://www.omim.org/entry/621297"},{"mim_id":"620989","title":"SERPIN FAMILY E, MEMBER 3; SERPINE3","url":"https://www.omim.org/entry/620989"},{"mim_id":"620957","title":"tRNA METHYLTRANSFERASE 10B; TRMT10B","url":"https://www.omim.org/entry/620957"},{"mim_id":"620738","title":"ZONE OF POLARIZING ACTIVITY REGULATORY SEQUENCE; ZRS","url":"https://www.omim.org/entry/620738"},{"mim_id":"620294","title":"CONGENITAL HEART DEFECTS, MULTIPLE TYPES, 9; CHTD9","url":"https://www.omim.org/entry/620294"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Vesicles","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":1138.3}],"url":"https://www.proteinatlas.org/search/CAT"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q6IB77","domains":[{"cath_id":"3.40.630","chopping":"2-132","consensus_level":"high","plddt":96.0507,"start":2,"end":132},{"cath_id":"3.40.630.30","chopping":"159-279","consensus_level":"high","plddt":96.703,"start":159,"end":279}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IB77","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IB77-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IB77-F1-predicted_aligned_error_v6.png","plddt_mean":95.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CAT","jax_strain_url":"https://www.jax.org/strain/search?query=CAT"},"sequence":{"accession":"Q6IB77","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6IB77.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6IB77/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IB77"}},"corpus_meta":[{"pmid":"457678","id":"PMC_457678","title":"Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. 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Some properties of the enzyme in rat and rabbit.","date":"1978","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/666745","citation_count":32,"is_preprint":false},{"pmid":"889778","id":"PMC_889778","title":"Photoaffinity labeling of acyl-coenzyme A:glycine N-acyltransferase with p-azidobenzoyl-coenzyme A.","date":"1977","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/889778","citation_count":31,"is_preprint":false},{"pmid":"27016726","id":"PMC_27016726","title":"Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells.","date":"2016","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/27016726","citation_count":23,"is_preprint":false},{"pmid":"26149650","id":"PMC_26149650","title":"Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway.","date":"2015","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26149650","citation_count":23,"is_preprint":false},{"pmid":"23237781","id":"PMC_23237781","title":"Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase.","date":"2012","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/23237781","citation_count":21,"is_preprint":false},{"pmid":"22408254","id":"PMC_22408254","title":"Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules.","date":"2012","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22408254","citation_count":16,"is_preprint":false},{"pmid":"9202629","id":"PMC_9202629","title":"Developmental profile of mitochondrial glycine N-acyltransferase in human liver.","date":"1997","source":"The Journal of pediatrics","url":"https://pubmed.ncbi.nlm.nih.gov/9202629","citation_count":15,"is_preprint":false},{"pmid":"22071172","id":"PMC_22071172","title":"Enzymatic characterization and elucidation of the catalytic mechanism of a recombinant bovine glycine N-acyltransferase.","date":"2011","source":"Drug metabolism and disposition: the biological fate of chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/22071172","citation_count":15,"is_preprint":false},{"pmid":"20925583","id":"PMC_20925583","title":"Genetic polymorphisms of glycine N-acyltransferase (GLYAT) in a French Caucasian population.","date":"2010","source":"Xenobiotica; the fate of foreign compounds in biological systems","url":"https://pubmed.ncbi.nlm.nih.gov/20925583","citation_count":14,"is_preprint":false},{"pmid":"31376196","id":"PMC_31376196","title":"Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer.","date":"2019","source":"The Prostate","url":"https://pubmed.ncbi.nlm.nih.gov/31376196","citation_count":13,"is_preprint":false},{"pmid":"19252341","id":"PMC_19252341","title":"Genetic polymorphisms of glycine N-acyltransferase in Japanese individuals.","date":"2009","source":"Drug metabolism and pharmacokinetics","url":"https://pubmed.ncbi.nlm.nih.gov/19252341","citation_count":13,"is_preprint":false},{"pmid":"24576660","id":"PMC_24576660","title":"Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli.","date":"2014","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/24576660","citation_count":12,"is_preprint":false},{"pmid":"33803916","id":"PMC_33803916","title":"Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl-CoA.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33803916","citation_count":10,"is_preprint":false},{"pmid":"33567294","id":"PMC_33567294","title":"Frequent sequence variants of human glycine N-acyltransferase (GLYAT) and inborn errors of metabolism.","date":"2021","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/33567294","citation_count":9,"is_preprint":false},{"pmid":"28759163","id":"PMC_28759163","title":"New insights into the catalytic mechanism of human glycine N-acyltransferase.","date":"2017","source":"Journal of biochemical and molecular toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/28759163","citation_count":7,"is_preprint":false},{"pmid":"38066746","id":"PMC_38066746","title":"Downregulation of Glycine N-Acyltransferase in Kidney Renal Clear Cell Carcinoma: A Bioinformatic-Based Screening.","date":"2023","source":"Diagnostics (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/38066746","citation_count":5,"is_preprint":false},{"pmid":"40288509","id":"PMC_40288509","title":"Glycine N-acyltransferase deficiency in sensory neurons suppresses osteoarthritis pain.","date":"2025","source":"The journal of pain","url":"https://pubmed.ncbi.nlm.nih.gov/40288509","citation_count":4,"is_preprint":false},{"pmid":"40747359","id":"PMC_40747359","title":"Glycine N-Acyltransferase Deficiency due to a Homozygous Nonsense Variant in the GLYAT: A Novel Inborn Error of Metabolism.","date":"2025","source":"JIMD reports","url":"https://pubmed.ncbi.nlm.nih.gov/40747359","citation_count":1,"is_preprint":false},{"pmid":"40938199","id":"PMC_40938199","title":"Structure of Bovine Glycine N-Acyltransferase Clarifies Its Catalytic Mechanism.","date":"2025","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/40938199","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"sonnet_model":"claude-sonnet-4-6","sonnet_input_tokens":14663,"sonnet_output_tokens":3483,"sonnet_usd":0.0962,"sonnet_elapsed_seconds":69.2,"stage1_stop_reason":"end_turn"},"stage2":{"opus_model":"claude-opus-4-8","opus_input_tokens":11180,"opus_output_tokens":3440,"opus_usd":0.1419,"opus_elapsed_seconds":36.7,"stage2_stop_reason":"end_turn"},"total_usd":0.2381},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1979,\n      \"finding\": \"Two distinct acyl-CoA:glycine N-acyltransferases were purified to near-homogeneity from bovine liver mitochondria, each consisting of a single polypeptide chain (~33 kDa). Preliminary kinetic studies were consistent with a sequential (ordered) reaction mechanism in which acyl-CoA binds first, glycine adds before CoA leaves, and the peptide product dissociates last.\",\n      \"method\": \"Enzyme purification, substrate specificity assays, stoichiometric cleavage assays, mass spectrometry of products, kinetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — near-homogeneity purification, stoichiometric product assays, kinetic mechanism established; replicated across two enzymes in same study\",\n      \"pmids\": [\"457678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1977,\n      \"finding\": \"Acyl-CoA:glycine N-acyltransferase from beef liver is composed of a single polypeptide (~35 kDa) with one active site per molecule, as demonstrated by photoaffinity labeling with p-azidobenzoyl-CoA. Benzoyl-CoA protects the enzyme from photoinactivation, confirming that p-azidobenzoyl-CoA targets the acyl-CoA binding active site.\",\n      \"method\": \"Photoaffinity labeling, UV irradiation, competitive protection assay with benzoyl-CoA\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site-directed photoaffinity labeling with competitive protection; single lab but multiple orthogonal methods (alternate substrate activity, photolabeling, protection)\",\n      \"pmids\": [\"889778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Acyl-CoA:glycine N-acyltransferase is exclusively an intramitochondrial enzyme in rat liver; no activity was detected in peroxisomes despite their capacity to produce acyl-CoAs.\",\n      \"method\": \"Subcellular fractionation, organelle-specific enzyme activity assays\",\n      \"journal\": \"Biochemical medicine and metabolic biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation with activity measurement; single lab but direct localization experiment\",\n      \"pmids\": [\"3741707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Human mitochondrial acyl-CoA:glycine N-acyltransferase (ACGNAT) was purified to homogeneity from adult liver; it is a monomer of 30 kDa (pI 6.8). Km values for benzoyl-CoA, salicyl-CoA, isovaleryl-CoA, and octanoyl-CoA were determined, establishing the enzyme's substrate preference profile.\",\n      \"method\": \"Protein purification to homogeneity, SDS-PAGE, isoelectric focusing, enzyme kinetics\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — purification to homogeneity with biochemical characterization; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"7802672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"N-fatty acylglycines are enzymatically produced from fatty acyl-CoA and glycine by acyl-CoA:glycine N-acyltransferase, and N-myristoylglycine is a substrate for peptidylglycine alpha-amidating enzyme, establishing a two-enzyme biosynthetic route to fatty acid primary amides.\",\n      \"method\": \"In vitro enzyme assay with N-myristoylglycine substrate, measurement of (V/K)app\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro enzyme assay establishing substrate activity; single lab, single method for the GLYAT step\",\n      \"pmids\": [\"8660675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The catalytic glutamate residue (Glu226 in bovine GLYAT) was identified as essential for activity: an E226Q mutant showed decreased activity at pH 8.0 that could be compensated by increasing pH, indicating Glu226 deprotonates glycine to facilitate nucleophilic attack on the acyl-CoA.\",\n      \"method\": \"Recombinant expression, E226Q active-site mutagenesis, enzyme kinetics at varying pH, protein modeling\",\n      \"journal\": \"Drug metabolism and disposition\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis combined with pH-rescue kinetics and molecular modeling; single lab but multiple orthogonal methods supporting the catalytic mechanism\",\n      \"pmids\": [\"22071172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Single nucleotide polymorphisms in human GLYAT alter enzyme activity: N156S variant is more active, F168L is less active, and R199C is completely inactive. An E227Q active-site mutant is also inactive, confirming Glu227 as the catalytic residue in human GLYAT. The R199C inactivity is attributed to destabilization of an α-loop-α motif important for substrate binding in the GNAT superfamily.\",\n      \"method\": \"Site-directed mutagenesis of recombinant human GLYAT, expression, purification, enzymatic characterization, molecular modeling with CoA\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis (E227Q) confirming catalytic residue, SNP variant functional characterization, structural modeling; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"23237781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human GLYAT is expressed specifically in liver and kidney, and the protein localizes to mitochondria, as confirmed by immunolabeling.\",\n      \"method\": \"Tissue-specific expression analysis, cellular immunolocalization with affinity-purified anti-GLYAT antibody\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct immunolocalization experiment with affinity-purified antibody; single lab, consistent with prior biochemical fractionation data\",\n      \"pmids\": [\"22475485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Human GLYAT exhibits mechanistic kinetic cooperativity (sigmoidal substrate activation with glycine variable and substrate inhibition with benzoyl-CoA variable) consistent with a Ferdinand enzyme mechanism, rather than the previously assumed Michaelis-Menten or ping-pong mechanism.\",\n      \"method\": \"Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods, allosteric sigmoidal enzyme kinetic modeling\",\n      \"journal\": \"Journal of biochemical and molecular toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two orthogonal analytical methods; single lab; contradicts prior mechanistic models\",\n      \"pmids\": [\"28759163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Human GLYAT wild-type protein localizes to the mitochondrial matrix when expressed as an eGFP fusion in HEK293 cells, confirming its intramitochondrial localization in a human cellular context. The p.(Gln61Leu) variant shows decreased specific activity and the p.(Asn156Ser) variant shows somewhat increased activity compared to wild-type.\",\n      \"method\": \"Recombinant expression in E. coli and HEK293 cells, eGFP fusion localization imaging, enzyme activity assay after purification\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell fluorescence imaging for localization plus enzymatic activity characterization; single lab, two orthogonal approaches\",\n      \"pmids\": [\"33567294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GLYAT exhibits cooperative substrate binding (two-substrate Hill kinetics) and the rare 156Asn>Ser,199Arg>Cys haplotype reduces kcat to ~9.8% of the most abundant haplotype while increasing affinity for benzoyl-CoA, demonstrating that Arg199 is critical for catalytic turnover.\",\n      \"method\": \"Recombinant expression of haplotype variants, kinetic characterization fitted to two-substrate Hill equation\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme kinetics with multiple haplotype variants; single lab, consistent with prior mutagenesis data on R199C\",\n      \"pmids\": [\"33803916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The first crystal structure of bovine GLYAT was solved in apo form and bound to benzoyl-CoA. Structural analysis and mutagenesis of key residues revealed that catalysis proceeds via a general base mechanism driven by a potential low-barrier hydrogen bond (LBHB) formed between a catalytic Glu-His dyad, with Glu deprotonating the glycine amine for nucleophilic attack on the acyl-CoA thioester.\",\n      \"method\": \"X-ray crystallography (apo and benzoyl-CoA-bound structures), active-site mutagenesis, functional validation\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determination with bound substrate combined with mutagenesis of catalytic residues; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"40938199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GLYAT is upregulated in dorsal root ganglion (DRG) neurons in a mouse osteoarthritis pain model. GLYAT overexpression increases reactive oxygen species (ROS) levels in DRG neurons and upregulates TRPV1, causing mechanical and thermal hyperalgesia; GLYAT loss-of-function alleviates pain-like behaviors. ROS clearance or TRPV1 blockade in GLYAT-overexpressing mice rescues hyperalgesia, placing GLYAT upstream of ROS→TRPV1 in pain signaling.\",\n      \"method\": \"Transcriptomic sequencing of DRG, viral overexpression and genetic knockout in mice, ROS measurement, TRPV1 immunolabeling, pharmacological rescue with ROS scavenger and TRPV1 blocker, behavioral nociception assays\",\n      \"journal\": \"The journal of pain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic gain- and loss-of-function with pharmacological rescue establishing pathway position; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"40288509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A homozygous nonsense variant (p.Q108Ter) in human GLYAT abolishes GLYAT enzymatic activity in vitro, causing glycine N-acyltransferase deficiency associated with developmental delays, confirming that GLYAT enzymatic activity is required for normal glycine conjugation metabolism in humans.\",\n      \"method\": \"Whole exome sequencing, Sanger confirmation, in vitro activity assay of the nonsense variant\",\n      \"journal\": \"JIMD reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single patient, in vitro activity abolishment confirmed; establishes loss-of-function consequence but single case\",\n      \"pmids\": [\"40747359\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Glycine N-acyltransferase (GLYAT) is a mitochondrial matrix enzyme (monomeric, ~30–35 kDa) that catalyzes the transfer of acyl groups from acyl-CoA donors (preferentially benzoyl-CoA and short/medium-chain acyl-CoAs) to glycine via a general base mechanism in which a catalytic Glu-His dyad (with a potential low-barrier hydrogen bond) deprotonates glycine to enable nucleophilic attack on the acyl-CoA thioester, releasing CoA and an N-acylglycine product; the enzyme displays cooperative (Ferdinand) kinetics, harbors a single active site per polypeptide, and its activity is modulated by key residues (Glu226/227, Arg199) whose mutation abolishes or severely reduces catalysis, while loss of GLYAT activity in vivo disrupts CoA homeostasis, glycine metabolism, and—in DRG neurons—promotes ROS accumulation and TRPV1 upregulation to drive pain sensitization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GLYAT (acyl-CoA:glycine N-acyltransferase) is a mitochondrial matrix enzyme of liver and kidney that conjugates glycine to acyl-CoA donors, generating N-acylglycine products as a route for detoxification of benzoate and short/medium-chain acyl groups [#3, #7, #9]. The purified enzyme is a single-chain monomer of ~30–35 kDa bearing one active site per polypeptide, with a preference for benzoyl-CoA and additional activity toward salicyl-, isovaleryl-, octanoyl-, and fatty acyl-CoA substrates [#0, #1, #3]; by acting on fatty acyl-CoA it feeds a two-enzyme route to fatty acid primary amides via downstream peptidylglycine alpha-amidating enzyme [#4]. Catalysis proceeds through a general base mechanism in which a catalytic glutamate (Glu226/Glu227) deprotonates the glycine amine to enable nucleophilic attack on the acyl-CoA thioester, a mechanism supported by pH-rescue of an E226Q/E227Q mutant and by the crystal structure of the enzyme bound to benzoyl-CoA revealing a Glu-His catalytic dyad [#5, #6, #11]; Arg199 is essential for catalytic turnover, as its loss (R199C) abolishes activity by destabilizing a substrate-binding motif [#6, #10]. Rather than simple Michaelis-Menten or ping-pong behavior, GLYAT displays cooperative (Ferdinand-type) kinetics with sigmoidal glycine activation and benzoyl-CoA substrate inhibition [#8, #10]. Loss-of-function consequences are established in vivo: a homozygous nonsense variant (p.Q108Ter) abolishes activity and causes glycine N-acyltransferase deficiency with developmental delays in humans [#13], and GLYAT upregulation in dorsal root ganglion neurons drives a ROS→TRPV1 axis that produces pain hypersensitivity in a mouse osteoarthritis model [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1979,\n      \"claim\": \"Established that glycine conjugation is carried out by discrete acyl-CoA:glycine N-acyltransferase enzymes with a defined ordered kinetic mechanism, settling the basic identity and reaction logic of the activity.\",\n      \"evidence\": \"Purification to near-homogeneity from bovine liver mitochondria with kinetic mechanism analysis\",\n      \"pmids\": [\"457678\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetic model later revised to cooperative rather than simple ordered\", \"No structural basis for catalysis defined\", \"Human enzyme not yet characterized\"]\n    },\n    {\n      \"year\": 1977,\n      \"claim\": \"Demonstrated the enzyme is a single polypeptide with one acyl-CoA binding active site, defining its architecture and the location of the catalytic site.\",\n      \"evidence\": \"Photoaffinity labeling with p-azidobenzoyl-CoA plus competitive protection by benzoyl-CoA in beef liver enzyme\",\n      \"pmids\": [\"889778\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No residue-level identification of catalytic groups\", \"No structure\"]\n    },\n    {\n      \"year\": 1986,\n      \"claim\": \"Resolved the subcellular compartment of the activity by showing it is exclusively intramitochondrial, distinguishing it from peroxisomal acyl-CoA metabolism.\",\n      \"evidence\": \"Subcellular fractionation with organelle-specific activity assays in rat liver\",\n      \"pmids\": [\"3741707\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Submitochondrial location not pinpointed\", \"Human localization not yet shown\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Characterized the human liver enzyme and its substrate preference profile, anchoring later human genetic and mechanistic work to a defined monomeric protein.\",\n      \"evidence\": \"Purification to homogeneity and kinetic determination of Km for benzoyl-, salicyl-, isovaleryl-, and octanoyl-CoA from human liver\",\n      \"pmids\": [\"7802672\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic residues not identified\", \"Gene/sequence-level variant effects unaddressed\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Extended the enzyme's biological scope by showing it produces N-fatty acylglycines that feed a two-enzyme pathway to fatty acid primary amides.\",\n      \"evidence\": \"In vitro enzyme assay with N-myristoylglycine and downstream peptidylglycine alpha-amidating enzyme\",\n      \"pmids\": [\"8660675\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological flux through this route not quantified\", \"Single method for the GLYAT step\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified the catalytic glutamate (Glu226/Glu227) and showed it acts as a general base, and demonstrated that natural human SNPs including the inactivating R199C alter activity, linking genotype to enzyme function.\",\n      \"evidence\": \"Active-site mutagenesis with pH-rescue kinetics, SNP variant characterization, and molecular modeling of recombinant bovine and human GLYAT\",\n      \"pmids\": [\"22071172\", \"23237781\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Catalytic partner (His) of the dyad not yet defined\", \"No experimental structure\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Confirmed tissue-restricted expression (liver, kidney) and mitochondrial localization of the human protein by direct immunolabeling.\",\n      \"evidence\": \"Tissue expression analysis and immunolocalization with affinity-purified anti-GLYAT antibody\",\n      \"pmids\": [\"22475485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Submitochondrial compartment not resolved by this method\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revised the kinetic model, showing GLYAT follows a cooperative Ferdinand mechanism rather than Michaelis-Menten or ping-pong, reframing how its activity is regulated by substrate.\",\n      \"evidence\": \"Bi-substrate kinetic analysis by colorimetric and HPLC-ESI-MS/MS methods with allosteric modeling\",\n      \"pmids\": [\"28759163\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of cooperativity unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Confirmed mitochondrial matrix localization in a human cellular context and quantified variant/haplotype effects, establishing Arg199 as critical for catalytic turnover.\",\n      \"evidence\": \"eGFP-fusion live-cell imaging in HEK293 and kinetic characterization of haplotype variants fitted to two-substrate Hill equations\",\n      \"pmids\": [\"33567294\", \"33803916\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which R199 supports turnover inferred from modeling, not structure\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the first atomic structure and definitive mechanism by capturing the benzoyl-CoA-bound enzyme and showing catalysis via a Glu-His dyad with a potential low-barrier hydrogen bond.\",\n      \"evidence\": \"X-ray crystallography of apo and benzoyl-CoA-bound bovine GLYAT with active-site mutagenesis\",\n      \"pmids\": [\"40938199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of human enzyme not solved\", \"Structural basis of cooperative kinetics not directly addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established physiological loss- and gain-of-function consequences: a nonsense variant causes human GLYAT deficiency with developmental delays, and GLYAT upregulation drives ROS→TRPV1 pain signaling in DRG neurons.\",\n      \"evidence\": \"Human exome sequencing with in vitro activity assay; mouse DRG overexpression/knockout with ROS and TRPV1 readouts and pharmacological rescue\",\n      \"pmids\": [\"40747359\", \"40288509\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-patient disease evidence\", \"Molecular link between GLYAT enzymatic output and ROS generation not defined\", \"Pain phenotype shown in mouse only\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GLYAT enzymatic activity mechanistically generates ROS and engages TRPV1, and how its cooperative kinetics are structurally encoded, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical link between acyl-CoA/glycine conjugation flux and ROS production\", \"No structure rationalizing Ferdinand cooperativity\", \"Human enzyme structure unsolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6, 11]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2, 7, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}","audit_flag":{"gene":"CAT","tier":"IDENTITY","verdict":"Identity concern","subtype":"corpus_ungrounded","uniprot_band":"medium","rules_fired":"R1,R6","issue":"R1: gene named in 0/26 (0%) of its own corpus abstracts (< 25%) — corpus likely a paralog/alias collision; R6: narrative-cited PMIDs vs gene2pubmed overlap = 0.00% (n_cited=14, n_g2p=488)"},"evaluation":{"pairwise":"win"}}