{"gene":"MCAT","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1992,"finding":"The E. coli fabD gene encodes malonyl-CoA:acyl carrier protein transacylase (MCT/FabD); cloning by complementation of a thermosensitive fabD mutant and overexpression yielded ~1000-fold increase in MCT activity, confirming fabD as the sole gene responsible for this transacylase activity. DNA sequence analysis revealed fabD is part of an operon with at least two other fatty acid biosynthesis genes (a ketoacyl-ACP synthase upstream and a ketoacyl-ACP reductase downstream).","method":"Genetic complementation of temperature-sensitive mutant, overexpression, enzyme activity assay, DNA sequencing, Western blot","journal":"Journal of bacteriology","confidence":"High","confidence_rationale":"Tier 1 / Strong — complementation, enzymatic activity confirmation, and operon structure established in a single rigorous study with multiple orthogonal methods","pmids":["1314802"],"is_preprint":false},{"year":1975,"finding":"The fabD locus encoding malonyl-CoA:acyl carrier protein transacylase in E. coli was mapped to minute 24 on the chromosome, between pyrC and purB and close to cat, by conjugation and P1 transduction. fabD mutants carry a thermolabile transacylase causing defective fatty acid synthesis and temperature-sensitive growth.","method":"Bacterial conjugation, P1 transduction genetic mapping, temperature-sensitive growth phenotype","journal":"Journal of bacteriology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — classical genetic mapping with two orthogonal methods (conjugation + transduction), single study","pmids":["1090584"],"is_preprint":false},{"year":1999,"finding":"Pseudomonas aeruginosa FabD (malonyl-CoA:ACP transacylase) was purified as a His-tagged fusion protein and shown in vitro to catalyze malonyl-CoA:ACP transacylase activity, using its cognate holo-ACP as acyl acceptor. A chromosomal temperature-sensitive fabD mutant (W258Q substitution) was generated by site-directed mutagenesis, demonstrating the essentiality of fabD for fatty acid biosynthesis.","method":"Protein purification, in vitro transacylase activity assay, site-directed mutagenesis, chromosomal mutant construction","journal":"Journal of bacteriology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic reconstitution combined with mutagenesis establishing catalytic requirement of the residue, single lab","pmids":["10464226"],"is_preprint":false},{"year":2003,"finding":"A continuous coupled enzyme assay for FabD (malonyl-CoA:ACP transacylase) was developed and validated, demonstrating that the CoASH product of the transacylation reaction can be coupled to alpha-ketoglutarate dehydrogenase (KDH) to monitor NAD+ reduction fluorometrically, confirming the mechanistic step of malonyl transfer from malonyl-CoA to the phosphopantetheine thiol of ACP with release of free CoASH.","method":"Coupled in vitro enzyme assay, fluorometric detection, microtiter plate reader","journal":"Analytical biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro enzymatic assay confirming reaction mechanism, but single lab and no mutagenesis","pmids":["12842120"],"is_preprint":false},{"year":2002,"finding":"Streptomyces glaucescens FabD (malonyl-CoA:ACP transacylase) was shown by in vitro kinetic analysis to have relaxed ACP specificity: it is active with both fatty acid synthase ACPs (FabC, AcpP) and polyketide synthase ACP (TcmM), with kcat values of 560, 141, and 150 min-1 and Km values of ~12-13 µM, respectively. This broad ACP specificity supports a role for FabD in providing malonyl-ACP precursors for both fatty acid and polyketide biosynthesis, in contrast to FabH which showed strict ACP specificity restricted to FAS ACPs.","method":"Purified recombinant protein, in vitro kinetic assay with multiple ACP substrates, isotope labeling","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro kinetics with multiple substrates and isotope labeling in one rigorous study establishing substrate specificity","pmids":["12173933"],"is_preprint":false},{"year":2006,"finding":"Crystal structures of E. coli FabD (malonyl-CoA:ACP transacylase) in binary complex with its natural substrate malonyl-CoA and other ligands revealed the active site architecture. The highly conserved residues Gln11, Ser92, Arg117, and His201 were identified as functionally critical: Ser92 acts as the nucleophile, Arg117 stabilizes the malonyl carboxylate, His201 acts as a general base/acid, and a preformed oxyanion hole stabilizes the transition state. A new catalytic mechanism was proposed based on the structural data.","method":"X-ray crystallography of binary complexes, structure-based active site analysis","journal":"Acta crystallographica. Section D, Biological crystallography","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures of enzyme-substrate complexes with detailed active site residue identification; mechanistic inference from structure without mutagenesis validation in this paper","pmids":["16699188"],"is_preprint":false},{"year":2020,"finding":"The X-ray crystal structure of E. coli FabD (malonyl-CoA:ACP transacylase) in covalent complex with its cognate ACP (AcpP), trapped by a crosslinking probe, revealed the type II FAS ACP-AT protein-protein interface. Mutational, kinetic, and long-timescale molecular dynamics analyses showed that AcpP adopts multiple productive conformations at the FabD binding interface (interfacial plasticity), enabling high transacylation rates. Rigid body subdomain motions within FabD were also identified as key to AT activity and substrate selectivity.","method":"Covalent crosslinking, X-ray crystallography of ACP-AT complex, site-directed mutagenesis, kinetic analysis, molecular dynamics simulation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of trapped complex combined with mutagenesis, kinetic analysis, and MD simulation providing orthogonal mechanistic support in one study","pmids":["32929027"],"is_preprint":false},{"year":2021,"finding":"Pathological variants in the human nuclear MCAT gene, encoding the mitochondrial malonyl-CoA:acyl carrier protein transacylase involved in fatty acid biogenesis, cause hereditary optic neuropathy with a LHON-like phenotype. Two novel MCAT mutations were identified in a patient with acute bilateral central visual loss, expanding the phenotypic spectrum of MCAT mutations beyond the previously reported childhood-onset insidious optic nerve degeneration.","method":"Molecular genetic analysis (sequencing) of patient cohort, clinical phenotype correlation","journal":"Genes","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic identification of disease-causing variants with phenotypic description but no direct biochemical/functional mechanistic experiment on MCAT protein function","pmids":["33918393"],"is_preprint":false}],"current_model":"MCAT/FabD is a malonyl-CoA:acyl carrier protein transacylase that catalyzes transfer of the malonyl moiety from malonyl-CoA to the phosphopantetheine thiol of holo-ACP (generating malonyl-ACP and free CoASH), the committed step providing the key extender unit for de novo fatty acid biosynthesis; its active site employs a Ser92 nucleophile, Arg117 for malonyl carboxylate stabilization, His201 as general base/acid, and a preformed oxyanion hole, and the enzyme accommodates multiple ACP partners via an interfacial plasticity mechanism that allows AcpP to adopt multiple productive conformations, sustaining high catalytic rates; in humans, loss-of-function MCAT mutations cause mitochondrial fatty acid synthesis deficiency manifesting as hereditary optic neuropathy."},"narrative":{"mechanistic_narrative":"MCAT (FabD) is a malonyl-CoA:acyl carrier protein transacylase that catalyzes the committed extender-unit step of de novo fatty acid biosynthesis, transferring the malonyl moiety from malonyl-CoA onto the phosphopantetheine thiol of holo-ACP to generate malonyl-ACP and free CoASH [PMID:1314802, PMID:12842120]. In bacteria the enzyme is encoded by a single essential gene, originally defined through complementation of a thermosensitive fabD mutant and shown to be indispensable for fatty acid synthesis, with thermolabile or substituted alleles abolishing growth [PMID:1314802, PMID:10464226]. Catalysis proceeds through a Ser92 nucleophile, with Arg117 stabilizing the malonyl carboxylate, His201 serving as general base/acid, and a preformed oxyanion hole stabilizing the transition state [PMID:16699188]. The enzyme engages its cognate ACP through a defined protein-protein interface in which ACP samples multiple productive conformations (interfacial plasticity) and rigid-body subdomain motions within the transacylase support high catalytic rates and substrate selectivity [PMID:32929027]. Substrate specificity toward ACP is relaxed, with activity on both fatty acid synthase and polyketide synthase ACPs, allowing it to provision malonyl-ACP for both pathways [PMID:12173933]. In humans, pathogenic variants in the nuclear-encoded mitochondrial MCAT cause a hereditary, LHON-like optic neuropathy [PMID:33918393].","teleology":[{"year":1975,"claim":"Establishing where the transacylase activity is encoded was the first step; mapping fabD to a defined chromosomal locus tied a temperature-sensitive fatty-acid-synthesis defect to a single genetic determinant.","evidence":"Conjugation and P1 transduction mapping with temperature-sensitive growth phenotype in E. coli","pmids":["1090584"],"confidence":"Medium","gaps":["Did not isolate or sequence the gene product","No biochemical demonstration of the encoded enzyme activity"]},{"year":1992,"claim":"Cloning fabD by complementation and overexpression proved it is the sole gene responsible for malonyl-CoA:ACP transacylase activity and placed it within a fatty acid biosynthesis operon.","evidence":"Genetic complementation, overexpression (~1000-fold activity increase), enzyme assay, and DNA sequencing in E. coli","pmids":["1314802"],"confidence":"High","gaps":["No structural information on the active site","Catalytic residues not yet identified"]},{"year":1999,"claim":"Purification and in vitro reconstitution with cognate holo-ACP, plus a chromosomal temperature-sensitive substitution, confirmed the enzyme's transacylase function and its essentiality in a second organism.","evidence":"His-tagged protein purification, in vitro transacylase assay, and site-directed chromosomal mutant (W258Q) in Pseudomonas aeruginosa","pmids":["10464226"],"confidence":"High","gaps":["Mechanism of the W258Q defect not resolved at residue level","Active-site chemistry not addressed"]},{"year":2002,"claim":"Kinetic profiling against multiple ACPs answered whether the enzyme is dedicated to fatty acid synthesis, showing relaxed ACP specificity that allows it to supply malonyl-ACP to both FAS and polyketide pathways.","evidence":"Purified recombinant Streptomyces glaucescens FabD with in vitro kinetics on FabC, AcpP, and TcmM ACPs and isotope labeling","pmids":["12173933"],"confidence":"High","gaps":["Structural basis of relaxed ACP recognition not defined","Does not address specificity in mammalian/mitochondrial systems"]},{"year":2003,"claim":"A continuous coupled assay confirmed the chemical step directly, monitoring CoASH release as malonyl is transferred to the ACP phosphopantetheine thiol.","evidence":"Coupled in vitro enzyme assay linking CoASH product to alpha-ketoglutarate dehydrogenase with fluorometric NAD+ detection","pmids":["12842120"],"confidence":"Medium","gaps":["No mutagenesis to assign catalytic residues","Single-lab assay validation"]},{"year":2006,"claim":"Crystal structures with malonyl-CoA defined the active-site architecture, assigning Ser92, Arg117, His201, and an oxyanion hole to specific catalytic roles and yielding a mechanistic model.","evidence":"X-ray crystallography of E. coli FabD binary substrate complexes with structure-based active-site analysis","pmids":["16699188"],"confidence":"High","gaps":["Mechanistic roles inferred from structure without mutagenesis in this study","ACP-bound state not captured"]},{"year":2020,"claim":"Trapping the covalent transacylase-ACP complex resolved how the enzyme engages its protein substrate, revealing interfacial plasticity and subdomain motions that sustain high catalytic rates and selectivity.","evidence":"Covalent crosslinking, X-ray structure of the ACP-AT complex, mutagenesis, kinetics, and molecular dynamics in E. coli","pmids":["32929027"],"confidence":"High","gaps":["Generalizability of the interface to mitochondrial MCAT not tested","Dynamics under turnover conditions inferred from simulation"]},{"year":2021,"claim":"Linking the human enzyme to disease established a physiological consequence of MCAT loss-of-function, identifying variants that cause a LHON-like hereditary optic neuropathy.","evidence":"Sequencing and clinical phenotype correlation in a patient with acute bilateral central visual loss","pmids":["33918393"],"confidence":"Low","gaps":["No functional assay demonstrating the variants impair MCAT enzymatic activity","Mechanism linking mitochondrial fatty acid synthesis deficit to optic neuropathy not established"]},{"year":null,"claim":"How human mitochondrial MCAT dysfunction mechanistically produces tissue-specific optic nerve degeneration remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No biochemical characterization of human MCAT catalysis or ACP partner in the timeline","Connection between mitochondrial malonyl-ACP supply and neuronal vulnerability uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,3,4,5]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,6]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,4]}],"complexes":[],"partners":["ACPP","FABC","TCMM"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8IVS2","full_name":"Malonyl-CoA-acyl carrier protein transacylase, mitochondrial","aliases":["Mitochondrial malonyl CoA:ACP acyltransferase","Mitochondrial malonyltransferase","[Acyl-carrier-protein] malonyltransferase"],"length_aa":390,"mass_kda":43.0,"function":"Catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of the mitochondrial ACP protein (NDUFAB1) (PubMed:12882974, PubMed:19549604). This suggests the existence of the biosynthesis of fatty acids in mitochondria (PubMed:12882974). Also acts as a mitochondrial small ribosomal subunit (mt-SSU) assembly factor (PubMed:36482135)","subcellular_location":"Mitochondrion","url":"https://www.uniprot.org/uniprotkb/Q8IVS2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MCAT","classification":"Not Classified","n_dependent_lines":113,"n_total_lines":1208,"dependency_fraction":0.0935430463576159},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MCAT","total_profiled":1310},"omim":[{"mim_id":"620583","title":"OPTIC ATROPHY 15; OPA15","url":"https://www.omim.org/entry/620583"},{"mim_id":"618383","title":"NEURODEVELOPMENTAL DISORDER WITH PROGRESSIVE MOVEMENT ABNORMALITIES, COGNITIVE DECLINE, AND BRAIN ABNORMALITIES; NEDMCB","url":"https://www.omim.org/entry/618383"},{"mim_id":"618181","title":"ZINC FINGER- AND BTB DOMAIN-CONTAINING PROTEIN 11; ZBTB11","url":"https://www.omim.org/entry/618181"},{"mim_id":"614479","title":"MALONYL CoA:ACP ACYLTRANSFERASE, MITOCHONDRIAL; MCAT","url":"https://www.omim.org/entry/614479"},{"mim_id":"609060","title":"COMBINED OXIDATIVE PHOSPHORYLATION DEFICIENCY 1; COXPD1","url":"https://www.omim.org/entry/609060"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Mitochondria","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MCAT"},"hgnc":{"alias_symbol":["MT","MCT","fabD","FASN2C","NET62","MCT1"],"prev_symbol":[]},"alphafold":{"accession":"Q8IVS2","domains":[{"cath_id":"3.40.366.10","chopping":"60-188_272-373","consensus_level":"high","plddt":97.004,"start":60,"end":373},{"cath_id":"3.30.70.250","chopping":"191-264","consensus_level":"medium","plddt":98.5936,"start":191,"end":264}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVS2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVS2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IVS2-F1-predicted_aligned_error_v6.png","plddt_mean":87.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MCAT","jax_strain_url":"https://www.jax.org/strain/search?query=MCAT"},"sequence":{"accession":"Q8IVS2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IVS2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IVS2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IVS2"}},"corpus_meta":[{"pmid":"1631128","id":"PMC_1631128","title":"MT-III, 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LHON-like Optic Neuropathy.","date":"2021","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/33918393","citation_count":32,"is_preprint":false},{"pmid":"32042910","id":"PMC_32042910","title":"Expanding the molecular and phenotypic spectrum of truncating MT-ATP6 mutations.","date":"2020","source":"Neurology. 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alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33589652","citation_count":26,"is_preprint":false},{"pmid":"19520270","id":"PMC_19520270","title":"Rolandic mitochondrial encephalomyelopathy and MT-ND3 mutations.","date":"2009","source":"Pediatric neurology","url":"https://pubmed.ncbi.nlm.nih.gov/19520270","citation_count":26,"is_preprint":false},{"pmid":"35513474","id":"PMC_35513474","title":"Sophisticated expression responses of ZNT1 and MT in response to changes in the expression of ZIPs.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35513474","citation_count":25,"is_preprint":false},{"pmid":"31108968","id":"PMC_31108968","title":"Melatonin MT1 and MT2 Receptors Exhibit Distinct Effects in the Modulation of Body Temperature across the Light/Dark Cycle.","date":"2019","source":"International journal 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genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34976016","citation_count":23,"is_preprint":false},{"pmid":"32430930","id":"PMC_32430930","title":"Nociceptive responses in melatonin MT2 receptor knockout mice compared to MT1 and double MT1 /MT2 receptor knockout mice.","date":"2020","source":"Journal of pineal research","url":"https://pubmed.ncbi.nlm.nih.gov/32430930","citation_count":23,"is_preprint":false},{"pmid":"35765703","id":"PMC_35765703","title":"Aristolochic acid I induces proximal tubule injury through ROS/HMGB1/mt DNA mediated activation of TLRs.","date":"2022","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35765703","citation_count":22,"is_preprint":false},{"pmid":"37495165","id":"PMC_37495165","title":"Molecular understanding of ER-MT communication dysfunction during neurodegeneration.","date":"2023","source":"Mitochondrion","url":"https://pubmed.ncbi.nlm.nih.gov/37495165","citation_count":21,"is_preprint":false},{"pmid":"24102627","id":"PMC_24102627","title":"Polymorphisms in the MT-ATP6 and MT-CYB genes in in vitro fertilization failure.","date":"2013","source":"Mitochondrial DNA","url":"https://pubmed.ncbi.nlm.nih.gov/24102627","citation_count":21,"is_preprint":false},{"pmid":"35581596","id":"PMC_35581596","title":"UPRmt activation improves pathological alterations in cellular models of mitochondrial diseases.","date":"2022","source":"Orphanet journal of rare diseases","url":"https://pubmed.ncbi.nlm.nih.gov/35581596","citation_count":21,"is_preprint":false},{"pmid":"17645699","id":"PMC_17645699","title":"Expression of the melatonin receptor (MT) 1 in benign and malignant human bone tumors.","date":"2007","source":"Journal of pineal research","url":"https://pubmed.ncbi.nlm.nih.gov/17645699","citation_count":21,"is_preprint":false},{"pmid":"34241926","id":"PMC_34241926","title":"Emerging roles of MT-MMPs in embryonic development.","date":"2021","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/34241926","citation_count":20,"is_preprint":false},{"pmid":"18803828","id":"PMC_18803828","title":"Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein.","date":"2008","source":"Plant methods","url":"https://pubmed.ncbi.nlm.nih.gov/18803828","citation_count":19,"is_preprint":false},{"pmid":"21187474","id":"PMC_21187474","title":"Comparison of metallothionein (MT) and Ki-67 antigen expression in benign and malignant thyroid tumours.","date":"2010","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/21187474","citation_count":19,"is_preprint":false},{"pmid":"16308482","id":"PMC_16308482","title":"The role of MT in neurological disorders.","date":"2005","source":"Journal of Alzheimer's disease : JAD","url":"https://pubmed.ncbi.nlm.nih.gov/16308482","citation_count":19,"is_preprint":false},{"pmid":"39454503","id":"PMC_39454503","title":"m7G-modified mt-tRF3b-LeuTAA regulates mitophagy and metabolic reprogramming via SUMOylation of SIRT3 in chondrocytes.","date":"2024","source":"Biomaterials","url":"https://pubmed.ncbi.nlm.nih.gov/39454503","citation_count":18,"is_preprint":false},{"pmid":"29180055","id":"PMC_29180055","title":"UPRmt coordinates immunity to maintain mitochondrial homeostasis and animal fitness.","date":"2017","source":"Mitochondrion","url":"https://pubmed.ncbi.nlm.nih.gov/29180055","citation_count":18,"is_preprint":false},{"pmid":"36362661","id":"PMC_36362661","title":"Mitochondrial Cardiomyopathy: The Roles of mt-tRNA Mutations.","date":"2022","source":"Journal of clinical medicine","url":"https://pubmed.ncbi.nlm.nih.gov/36362661","citation_count":18,"is_preprint":false},{"pmid":"34635923","id":"PMC_34635923","title":"Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism.","date":"2022","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34635923","citation_count":18,"is_preprint":false},{"pmid":"28159659","id":"PMC_28159659","title":"Day/night expression of MT1 and MT2 receptors in hypothalamic nuclei of the primate Sapajus apella.","date":"2017","source":"Journal of chemical neuroanatomy","url":"https://pubmed.ncbi.nlm.nih.gov/28159659","citation_count":18,"is_preprint":false},{"pmid":"28366030","id":"PMC_28366030","title":"Genetic diversities of MT-ND1 and MT-ND2 genes are associated with high-altitude adaptation in yak.","date":"2017","source":"Mitochondrial DNA. Part A, DNA mapping, sequencing, and analysis","url":"https://pubmed.ncbi.nlm.nih.gov/28366030","citation_count":17,"is_preprint":false},{"pmid":"25149069","id":"PMC_25149069","title":"MT-Toolbox: improved amplicon sequencing using molecule tags.","date":"2014","source":"BMC bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/25149069","citation_count":17,"is_preprint":false},{"pmid":"37817014","id":"PMC_37817014","title":"Dual roles of UPRer and UPRmt in neurodegenerative diseases.","date":"2023","source":"Journal of molecular medicine (Berlin, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/37817014","citation_count":16,"is_preprint":false},{"pmid":"32953045","id":"PMC_32953045","title":"Genetic hitchhiking, mitonuclear coadaptation, and the origins of mt DNA barcode gaps.","date":"2020","source":"Ecology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/32953045","citation_count":16,"is_preprint":false},{"pmid":"29410206","id":"PMC_29410206","title":"Regulation of spermatid polarity by the actin- and microtubule (MT)-based cytoskeletons.","date":"2018","source":"Seminars in cell & developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/29410206","citation_count":15,"is_preprint":false},{"pmid":"17669619","id":"PMC_17669619","title":"Expression of ZnT-1 (Slc30a1) and MT-1 (Mt1) in the conceptus of cadmium treated mice.","date":"2007","source":"Reproductive toxicology (Elmsford, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/17669619","citation_count":15,"is_preprint":false},{"pmid":"32869280","id":"PMC_32869280","title":"Progressive external ophthalmoplegia associated with novel MT-TN mutations.","date":"2020","source":"Acta neurologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/32869280","citation_count":15,"is_preprint":false},{"pmid":"37312933","id":"PMC_37312933","title":"Cu3P/1-MT Nanocomposites Potentiated Photothermal-Immunotherapy.","date":"2023","source":"International journal of nanomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/37312933","citation_count":14,"is_preprint":false},{"pmid":"34877647","id":"PMC_34877647","title":"Genotype-phenotype analysis of MT-ATP6-associated Leigh syndrome.","date":"2021","source":"Acta neurologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/34877647","citation_count":14,"is_preprint":false},{"pmid":"33476484","id":"PMC_33476484","title":"Epilepsy in MT-ATP6 - related mils/NARP: correlation of elettroclinical features with heteroplasmy.","date":"2021","source":"Annals of clinical and translational neurology","url":"https://pubmed.ncbi.nlm.nih.gov/33476484","citation_count":14,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":46792,"output_tokens":2673,"usd":0.090235,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9514,"output_tokens":2734,"usd":0.05796,"stage2_stop_reason":"end_turn"},"total_usd":0.148195,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking through the abstracts, I need to identify papers about MCAT (malonyl-CoA-acyl carrier protein transacylase, also known as fabD, FASN2C, NET62) — the mitochondrial enzyme involved in fatty acid biosynthesis.\n\nMost papers in this corpus are about completely different genes/proteins sharing the \"MT\" abbreviation (metallothioneins, melatonin receptors, MT-MMPs, mitochondrial DNA genes, microtubules, etc.). I need to focus only on papers about MCAT/fabD.\n\nRelevant papers:\n- PMID 16699188: Crystal structure of E. coli FabD (malonyl-CoA:ACP transacylase) — ortholog\n- PMID 10464226: P. aeruginosa FabD characterization — ortholog\n- PMID 1314802: E. coli fabD gene cloning — ortholog\n- PMID 1090584: E. coli fabD mapping — ortholog\n- PMID 12842120: FabD enzyme assay — ortholog\n- PMID 12173933: Streptomyces FabD characterization — ortholog\n- PMID 32929027: E. coli FabD ACP-AT complex structure — ortholog\n- PMID 33918393: MCAT mutations causing optic neuropathy — human MCAT\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"The E. coli fabD gene encodes malonyl-CoA:acyl carrier protein transacylase (MCT/FabD); cloning by complementation of a thermosensitive fabD mutant and overexpression yielded ~1000-fold increase in MCT activity, confirming fabD as the sole gene responsible for this transacylase activity. DNA sequence analysis revealed fabD is part of an operon with at least two other fatty acid biosynthesis genes (a ketoacyl-ACP synthase upstream and a ketoacyl-ACP reductase downstream).\",\n      \"method\": \"Genetic complementation of temperature-sensitive mutant, overexpression, enzyme activity assay, DNA sequencing, Western blot\",\n      \"journal\": \"Journal of bacteriology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — complementation, enzymatic activity confirmation, and operon structure established in a single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"1314802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1975,\n      \"finding\": \"The fabD locus encoding malonyl-CoA:acyl carrier protein transacylase in E. coli was mapped to minute 24 on the chromosome, between pyrC and purB and close to cat, by conjugation and P1 transduction. fabD mutants carry a thermolabile transacylase causing defective fatty acid synthesis and temperature-sensitive growth.\",\n      \"method\": \"Bacterial conjugation, P1 transduction genetic mapping, temperature-sensitive growth phenotype\",\n      \"journal\": \"Journal of bacteriology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — classical genetic mapping with two orthogonal methods (conjugation + transduction), single study\",\n      \"pmids\": [\"1090584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Pseudomonas aeruginosa FabD (malonyl-CoA:ACP transacylase) was purified as a His-tagged fusion protein and shown in vitro to catalyze malonyl-CoA:ACP transacylase activity, using its cognate holo-ACP as acyl acceptor. A chromosomal temperature-sensitive fabD mutant (W258Q substitution) was generated by site-directed mutagenesis, demonstrating the essentiality of fabD for fatty acid biosynthesis.\",\n      \"method\": \"Protein purification, in vitro transacylase activity assay, site-directed mutagenesis, chromosomal mutant construction\",\n      \"journal\": \"Journal of bacteriology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic reconstitution combined with mutagenesis establishing catalytic requirement of the residue, single lab\",\n      \"pmids\": [\"10464226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"A continuous coupled enzyme assay for FabD (malonyl-CoA:ACP transacylase) was developed and validated, demonstrating that the CoASH product of the transacylation reaction can be coupled to alpha-ketoglutarate dehydrogenase (KDH) to monitor NAD+ reduction fluorometrically, confirming the mechanistic step of malonyl transfer from malonyl-CoA to the phosphopantetheine thiol of ACP with release of free CoASH.\",\n      \"method\": \"Coupled in vitro enzyme assay, fluorometric detection, microtiter plate reader\",\n      \"journal\": \"Analytical biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro enzymatic assay confirming reaction mechanism, but single lab and no mutagenesis\",\n      \"pmids\": [\"12842120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Streptomyces glaucescens FabD (malonyl-CoA:ACP transacylase) was shown by in vitro kinetic analysis to have relaxed ACP specificity: it is active with both fatty acid synthase ACPs (FabC, AcpP) and polyketide synthase ACP (TcmM), with kcat values of 560, 141, and 150 min-1 and Km values of ~12-13 µM, respectively. This broad ACP specificity supports a role for FabD in providing malonyl-ACP precursors for both fatty acid and polyketide biosynthesis, in contrast to FabH which showed strict ACP specificity restricted to FAS ACPs.\",\n      \"method\": \"Purified recombinant protein, in vitro kinetic assay with multiple ACP substrates, isotope labeling\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro kinetics with multiple substrates and isotope labeling in one rigorous study establishing substrate specificity\",\n      \"pmids\": [\"12173933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Crystal structures of E. coli FabD (malonyl-CoA:ACP transacylase) in binary complex with its natural substrate malonyl-CoA and other ligands revealed the active site architecture. The highly conserved residues Gln11, Ser92, Arg117, and His201 were identified as functionally critical: Ser92 acts as the nucleophile, Arg117 stabilizes the malonyl carboxylate, His201 acts as a general base/acid, and a preformed oxyanion hole stabilizes the transition state. A new catalytic mechanism was proposed based on the structural data.\",\n      \"method\": \"X-ray crystallography of binary complexes, structure-based active site analysis\",\n      \"journal\": \"Acta crystallographica. Section D, Biological crystallography\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures of enzyme-substrate complexes with detailed active site residue identification; mechanistic inference from structure without mutagenesis validation in this paper\",\n      \"pmids\": [\"16699188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The X-ray crystal structure of E. coli FabD (malonyl-CoA:ACP transacylase) in covalent complex with its cognate ACP (AcpP), trapped by a crosslinking probe, revealed the type II FAS ACP-AT protein-protein interface. Mutational, kinetic, and long-timescale molecular dynamics analyses showed that AcpP adopts multiple productive conformations at the FabD binding interface (interfacial plasticity), enabling high transacylation rates. Rigid body subdomain motions within FabD were also identified as key to AT activity and substrate selectivity.\",\n      \"method\": \"Covalent crosslinking, X-ray crystallography of ACP-AT complex, site-directed mutagenesis, kinetic analysis, molecular dynamics simulation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of trapped complex combined with mutagenesis, kinetic analysis, and MD simulation providing orthogonal mechanistic support in one study\",\n      \"pmids\": [\"32929027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pathological variants in the human nuclear MCAT gene, encoding the mitochondrial malonyl-CoA:acyl carrier protein transacylase involved in fatty acid biogenesis, cause hereditary optic neuropathy with a LHON-like phenotype. Two novel MCAT mutations were identified in a patient with acute bilateral central visual loss, expanding the phenotypic spectrum of MCAT mutations beyond the previously reported childhood-onset insidious optic nerve degeneration.\",\n      \"method\": \"Molecular genetic analysis (sequencing) of patient cohort, clinical phenotype correlation\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic identification of disease-causing variants with phenotypic description but no direct biochemical/functional mechanistic experiment on MCAT protein function\",\n      \"pmids\": [\"33918393\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MCAT/FabD is a malonyl-CoA:acyl carrier protein transacylase that catalyzes transfer of the malonyl moiety from malonyl-CoA to the phosphopantetheine thiol of holo-ACP (generating malonyl-ACP and free CoASH), the committed step providing the key extender unit for de novo fatty acid biosynthesis; its active site employs a Ser92 nucleophile, Arg117 for malonyl carboxylate stabilization, His201 as general base/acid, and a preformed oxyanion hole, and the enzyme accommodates multiple ACP partners via an interfacial plasticity mechanism that allows AcpP to adopt multiple productive conformations, sustaining high catalytic rates; in humans, loss-of-function MCAT mutations cause mitochondrial fatty acid synthesis deficiency manifesting as hereditary optic neuropathy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MCAT (FabD) is a malonyl-CoA:acyl carrier protein transacylase that catalyzes the committed extender-unit step of de novo fatty acid biosynthesis, transferring the malonyl moiety from malonyl-CoA onto the phosphopantetheine thiol of holo-ACP to generate malonyl-ACP and free CoASH [#0, #3]. In bacteria the enzyme is encoded by a single essential gene, originally defined through complementation of a thermosensitive fabD mutant and shown to be indispensable for fatty acid synthesis, with thermolabile or substituted alleles abolishing growth [#0, #2]. Catalysis proceeds through a Ser92 nucleophile, with Arg117 stabilizing the malonyl carboxylate, His201 serving as general base/acid, and a preformed oxyanion hole stabilizing the transition state [#5]. The enzyme engages its cognate ACP through a defined protein-protein interface in which ACP samples multiple productive conformations (interfacial plasticity) and rigid-body subdomain motions within the transacylase support high catalytic rates and substrate selectivity [#6]. Substrate specificity toward ACP is relaxed, with activity on both fatty acid synthase and polyketide synthase ACPs, allowing it to provision malonyl-ACP for both pathways [#4]. In humans, pathogenic variants in the nuclear-encoded mitochondrial MCAT cause a hereditary, LHON-like optic neuropathy [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1975,\n      \"claim\": \"Establishing where the transacylase activity is encoded was the first step; mapping fabD to a defined chromosomal locus tied a temperature-sensitive fatty-acid-synthesis defect to a single genetic determinant.\",\n      \"evidence\": \"Conjugation and P1 transduction mapping with temperature-sensitive growth phenotype in E. coli\",\n      \"pmids\": [\"1090584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not isolate or sequence the gene product\", \"No biochemical demonstration of the encoded enzyme activity\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Cloning fabD by complementation and overexpression proved it is the sole gene responsible for malonyl-CoA:ACP transacylase activity and placed it within a fatty acid biosynthesis operon.\",\n      \"evidence\": \"Genetic complementation, overexpression (~1000-fold activity increase), enzyme assay, and DNA sequencing in E. coli\",\n      \"pmids\": [\"1314802\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural information on the active site\", \"Catalytic residues not yet identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Purification and in vitro reconstitution with cognate holo-ACP, plus a chromosomal temperature-sensitive substitution, confirmed the enzyme's transacylase function and its essentiality in a second organism.\",\n      \"evidence\": \"His-tagged protein purification, in vitro transacylase assay, and site-directed chromosomal mutant (W258Q) in Pseudomonas aeruginosa\",\n      \"pmids\": [\"10464226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of the W258Q defect not resolved at residue level\", \"Active-site chemistry not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Kinetic profiling against multiple ACPs answered whether the enzyme is dedicated to fatty acid synthesis, showing relaxed ACP specificity that allows it to supply malonyl-ACP to both FAS and polyketide pathways.\",\n      \"evidence\": \"Purified recombinant Streptomyces glaucescens FabD with in vitro kinetics on FabC, AcpP, and TcmM ACPs and isotope labeling\",\n      \"pmids\": [\"12173933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of relaxed ACP recognition not defined\", \"Does not address specificity in mammalian/mitochondrial systems\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"A continuous coupled assay confirmed the chemical step directly, monitoring CoASH release as malonyl is transferred to the ACP phosphopantetheine thiol.\",\n      \"evidence\": \"Coupled in vitro enzyme assay linking CoASH product to alpha-ketoglutarate dehydrogenase with fluorometric NAD+ detection\",\n      \"pmids\": [\"12842120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis to assign catalytic residues\", \"Single-lab assay validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Crystal structures with malonyl-CoA defined the active-site architecture, assigning Ser92, Arg117, His201, and an oxyanion hole to specific catalytic roles and yielding a mechanistic model.\",\n      \"evidence\": \"X-ray crystallography of E. coli FabD binary substrate complexes with structure-based active-site analysis\",\n      \"pmids\": [\"16699188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic roles inferred from structure without mutagenesis in this study\", \"ACP-bound state not captured\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Trapping the covalent transacylase-ACP complex resolved how the enzyme engages its protein substrate, revealing interfacial plasticity and subdomain motions that sustain high catalytic rates and selectivity.\",\n      \"evidence\": \"Covalent crosslinking, X-ray structure of the ACP-AT complex, mutagenesis, kinetics, and molecular dynamics in E. coli\",\n      \"pmids\": [\"32929027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generalizability of the interface to mitochondrial MCAT not tested\", \"Dynamics under turnover conditions inferred from simulation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linking the human enzyme to disease established a physiological consequence of MCAT loss-of-function, identifying variants that cause a LHON-like hereditary optic neuropathy.\",\n      \"evidence\": \"Sequencing and clinical phenotype correlation in a patient with acute bilateral central visual loss\",\n      \"pmids\": [\"33918393\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional assay demonstrating the variants impair MCAT enzymatic activity\", \"Mechanism linking mitochondrial fatty acid synthesis deficit to optic neuropathy not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How human mitochondrial MCAT dysfunction mechanistically produces tissue-specific optic nerve degeneration remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No biochemical characterization of human MCAT catalysis or ACP partner in the timeline\", \"Connection between mitochondrial malonyl-ACP supply and neuronal vulnerability uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"AcpP\",\n      \"FabC\",\n      \"TcmM\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}