{"gene":"BHMT2","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2008,"finding":"Purified recombinant human BHMT2 is a zinc metalloenzyme that uses S-methylmethionine (SMM) as the methyl donor for methylation of homocysteine to methionine; it cannot use betaine (unlike BHMT). The Km for SMM is 0.94 mM, and turnover is similar to BHMT. Methionine is a stronger inhibitor of BHMT2 than BHMT; S-adenosylmethionine is a weak inhibitor of BHMT2 but does not inhibit BHMT; dimethylglycine and betaine do not inhibit BHMT2.","method":"In vitro enzymatic assay with purified recombinant protein; kinetic parameter determination; inhibitor profiling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution with purified recombinant enzyme, kinetic characterization, and substrate/inhibitor profiling in a single rigorous study","pmids":["18230605"],"is_preprint":false},{"year":2008,"finding":"BHMT2 protein expressed in mammalian (COS-1) cells is unstable and rapidly degraded in rabbit reticulocyte lysate, but can be stabilized by co-transfection with BHMT; after co-transfection BHMT2 co-precipitates with BHMT, indicating a physical interaction between the two proteins.","method":"Co-transfection of COS-1 cells, co-immunoprecipitation, rabbit reticulocyte lysate degradation assay","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-immunoprecipitation and degradation assay from a single lab, two complementary methods","pmids":["18457970"],"is_preprint":false},{"year":2009,"finding":"Mouse Bhmt2, acting through its substrate S-methylmethionine (SMM), protects against acetaminophen-induced liver toxicity in vivo by influencing methionine and glutathione biosynthesis; this protective effect is diet-dependent because SMM is only synthesized in plants.","method":"Integrative genomic analysis across multiple inbred mouse strains combined with NMR-based metabolomics; in vivo dietary manipulation","journal":"Genome research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multi-strain genetic analysis combined with metabolomics and in vivo dietary intervention, replicated across mouse strains","pmids":["19923254"],"is_preprint":false},{"year":2000,"finding":"The human BHMT2 gene encodes a predicted 363-amino-acid protein (40.3 kDa) with 73% amino acid identity to BHMT. BHMT2 mRNA is most abundant in adult liver and kidney, with reduced expression in brain, heart, and skeletal muscle.","method":"cDNA cloning, gene sequencing, physical mapping, Northern blot/expression analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — cDNA cloning and expression profiling from a single lab, multiple tissues examined","pmids":["11087663"],"is_preprint":false},{"year":2010,"finding":"Mouse liver extracts containing BHMT2 recognize (S,S)-AdoMet but NOT (R,S)-AdoMet, and no enzymatic breakdown of (R,S)-AdoMet was detected in these extracts, indicating BHMT2 does not metabolize the age-damaged AdoMet diastereomer.","method":"In vitro enzyme assay with mouse liver extracts; substrate specificity testing","journal":"Rejuvenation research","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct in vitro assay with native liver extracts, single lab, negative result regarding (R,S)-AdoMet as substrate","pmids":["20370499"],"is_preprint":false},{"year":2019,"finding":"In Bhmt knockout mice, Bhmt2 expression is upregulated in the cochlea following noise damage, suggesting a compensatory role for BHMT2 in cochlear methionine metabolism when BHMT is absent.","method":"Bhmt knockout mouse model; quantitative RT-PCR of cochlear gene expression; RNA arrays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function mouse model with transcriptional readout, single lab, compensatory upregulation inferred from gene expression data","pmids":["30753104"],"is_preprint":false},{"year":2015,"finding":"SPIC transcription factor binds to enhancer elements near Bhmt2 (and Bhmt, Dmgdh) in mouse embryonic stem cells and stabilizes NANOG binding, controlling betaine-dependent one-carbon metabolism including SMM-to-SAH flux and histone methylation marks (H3R17me2 and H3K4me3).","method":"ChIP-seq, gain-of-function and loss-of-function experiments in ESCs, metabolic flux analysis","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq binding data combined with gain/loss-of-function experiments and metabolic readout in a single lab study","pmids":["37595034"],"is_preprint":false},{"year":2025,"finding":"In intestinal epithelial cells during necrotizing enterocolitis, BHMT2 and MAT1A regulate AHSG expression through S-adenosylmethionine production and histone methylation; silencing BHMT2 in LPS-stimulated HPIECs attenuates M1 macrophage polarization, inflammatory cytokine production, and macrophage invasive capacity in a transwell co-culture system, while BHMT2 overexpression promotes M1 macrophage activation.","method":"siRNA knockdown and overexpression in human primary intestinal epithelial cells; transwell co-culture with THP-1 macrophages; chromatin immunoprecipitation; RT-qPCR; Western blotting; ELISA; flow cytometry; NEC mouse model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (ChIP, co-culture, in vivo mouse model, gain/loss of function), single lab","pmids":["41219226"],"is_preprint":false},{"year":2015,"finding":"Porcine BHMT2 protein (363 amino acids) shares 78% amino acid identity with porcine BHMT, and both genes have 8 exons spanning ~16 kb (BHMT2) and ~26 kb (BHMT). BHMT2 transcripts are most abundant in liver and kidney cortex in pigs, mirroring the human expression pattern; neither gene has a TATA or CAAT box but both have a CpG island at the promoter/transcriptional start site.","method":"cDNA cloning, RLM-RACE, gene structure analysis, qPCR expression profiling","journal":"Gene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — molecular characterization in a pig model, single lab, primarily structural/expression data","pmids":["21156199"],"is_preprint":false},{"year":2015,"finding":"Evolutionary analysis across 38 deuterostome species showed that BHMT2 arose by gene duplication from BHMT after divergence of mammals from other vertebrates, with accelerated evolutionary rates relative to BHMT; codons under positive selection map to enzymatic or oligomerization domains, suggesting involvement in enzyme function. Two deletions in BHMT2 relative to BHMT affect oligomerization and methyl donor specificity.","method":"Comparative genomics and evolutionary rate analysis (dN/dS, GA Branch analysis) across 38 species","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 4 / Moderate — computational/comparative genomic analysis, no direct biochemical experiment; replicated across many species but purely computational","pmids":["26213999"],"is_preprint":false}],"current_model":"BHMT2 is a mammalian zinc metalloenzyme that remethylates homocysteine to methionine using S-methylmethionine (SMM) — not betaine — as the methyl donor; it is expressed primarily in liver and kidney, is stabilized by physical interaction with BHMT, protects against acetaminophen-induced hepatotoxicity in a diet-dependent manner through effects on methionine and glutathione biosynthesis, and in intestinal epithelial cells regulates M1 macrophage activation via an SAM/histone methylation-dependent axis involving MAT1A and AHSG."},"narrative":{"mechanistic_narrative":"BHMT2 is a mammalian zinc metalloenzyme of hepatic and renal one-carbon metabolism that remethylates homocysteine to methionine, using S-methylmethionine (SMM) rather than betaine as its methyl donor — a substrate specificity that distinguishes it from its paralog BHMT [PMID:18230605, PMID:11087663]. Kinetic characterization of purified recombinant enzyme established an SMM Km of ~0.94 mM with turnover comparable to BHMT, and a distinct inhibitor profile in which methionine is a strong inhibitor and S-adenosylmethionine a weak one, while betaine and dimethylglycine do not inhibit [PMID:18230605]. The protein is intrinsically unstable in mammalian cells and is stabilized through direct physical interaction with BHMT, with which it co-precipitates [PMID:18457970]. Through its SMM-dependent contribution to methionine and downstream glutathione biosynthesis, BHMT2 protects against acetaminophen-induced hepatotoxicity in a diet-dependent manner, since SMM is supplied only by plant sources [PMID:19923254]. Beyond classical one-carbon metabolism, BHMT2 feeds S-adenosylmethionine production and histone methylation: acting with MAT1A in intestinal epithelial cells, it regulates AHSG expression and drives M1 macrophage activation and inflammatory cytokine output [PMID:41219226].","teleology":[{"year":2000,"claim":"Before its biochemistry was known, the question was whether the BHMT-like gene encoded a distinct, tissue-restricted enzyme; cloning defined a 363-residue protein highly similar to BHMT but expressed predominantly in liver and kidney.","evidence":"cDNA cloning, physical mapping and Northern expression profiling of human BHMT2","pmids":["11087663"],"confidence":"Medium","gaps":["No catalytic activity or substrate demonstrated","Subcellular localization not established"]},{"year":2008,"claim":"The central mechanistic question — what reaction BHMT2 catalyzes — was answered by showing it is a zinc metalloenzyme that remethylates homocysteine using SMM, not betaine, defining its functional divergence from BHMT.","evidence":"In vitro enzymatic assay with purified recombinant enzyme, kinetic and inhibitor profiling","pmids":["18230605"],"confidence":"High","gaps":["No crystal structure or catalytic-mechanism model","Physiological flux through the SMM pathway in vivo not quantified in this study"]},{"year":2008,"claim":"Why BHMT2 protein is hard to express was addressed by showing it is rapidly degraded but stabilized by BHMT, establishing a physical interaction with its paralog as a determinant of protein stability.","evidence":"Co-transfection in COS-1 cells, co-immunoprecipitation, reticulocyte lysate degradation assay","pmids":["18457970"],"confidence":"Medium","gaps":["Single lab, no reciprocal IP or stoichiometry of the BHMT/BHMT2 complex","Degradation pathway and ubiquitin involvement unknown"]},{"year":2009,"claim":"The physiological consequence of BHMT2 activity was tied to hepatoprotection: through SMM it influences methionine and glutathione biosynthesis to limit acetaminophen toxicity, with the effect gated by dietary SMM availability.","evidence":"Multi-strain mouse genetic analysis with NMR metabolomics and in vivo dietary manipulation","pmids":["19923254"],"confidence":"High","gaps":["Direct enzymatic step in vivo inferred rather than measured","Human dietary relevance not tested"]},{"year":2010,"claim":"Substrate-specificity boundaries were refined by testing AdoMet diastereomers, showing BHMT2-containing liver extracts handle (S,S)-AdoMet but do not metabolize the age-damaged (R,S)-AdoMet.","evidence":"In vitro assay with mouse liver extracts and diastereomer substrate testing","pmids":["20370499"],"confidence":"Medium","gaps":["Used crude extracts, not purified BHMT2","Negative result does not pinpoint the responsible enzyme"]},{"year":2019,"claim":"Whether BHMT2 can compensate for BHMT loss was addressed by showing its transcriptional upregulation in cochlea of Bhmt knockout mice after noise damage, implicating a backup role in tissue methionine metabolism.","evidence":"Bhmt knockout mouse, cochlear qRT-PCR and RNA arrays","pmids":["30753104"],"confidence":"Medium","gaps":["Compensation inferred from mRNA only, not protein or activity","Functional rescue not demonstrated"]},{"year":2015,"claim":"Transcriptional control of the SMM/one-carbon module was placed under SPIC, which binds Bhmt2 enhancers and stabilizes NANOG to govern SMM-to-SAH flux and histone methylation in embryonic stem cells.","evidence":"ChIP-seq with gain/loss-of-function and metabolic flux analysis in mouse ESCs","pmids":["37595034"],"confidence":"Medium","gaps":["Direct contribution of BHMT2 versus co-regulated Bhmt/Dmgdh not separated","Mechanism in differentiated tissues unknown"]},{"year":2025,"claim":"An immunometabolic role was established: in intestinal epithelial cells BHMT2 with MAT1A controls AHSG via SAM and histone methylation, and BHMT2 levels bidirectionally set M1 macrophage polarization and inflammatory output.","evidence":"siRNA/overexpression in primary intestinal epithelial cells, transwell co-culture with macrophages, ChIP, and NEC mouse model","pmids":["41219226"],"confidence":"Medium","gaps":["Single lab; causal chain from BHMT2 enzymatic activity to histone marks not fully reconstituted","Direct enzymatic versus structural contribution of BHMT2 not dissected"]},{"year":null,"claim":"It remains unresolved how BHMT2 catalysis is structurally organized and how its SMM-derived methyl flux is mechanistically linked to the SAM/histone-methylation signaling roles emerging in stem cells and immune contexts.","evidence":"","pmids":[],"confidence":"Low","gaps":["No experimental structure of BHMT2","No defined regulatory mechanism connecting metabolic activity to chromatin output","Human in vivo physiological role beyond mouse models uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]}],"localization":[],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[6,7]}],"complexes":[],"partners":["BHMT","MAT1A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H2M3","full_name":"S-methylmethionine--homocysteine S-methyltransferase BHMT2","aliases":["Betaine--homocysteine S-methyltransferase 2"],"length_aa":363,"mass_kda":40.4,"function":"Involved in the regulation of homocysteine metabolism. Converts homocysteine to methionine using S-methylmethionine (SMM) as a methyl donor","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9H2M3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BHMT2","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/BHMT2","total_profiled":1310},"omim":[{"mim_id":"605932","title":"BETAINE-HOMOCYSTEINE METHYLTRANSFERASE 2; BHMT2","url":"https://www.omim.org/entry/605932"},{"mim_id":"602888","title":"BETAINE-HOMOCYSTEINE METHYLTRANSFERASE; BHMT","url":"https://www.omim.org/entry/602888"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"kidney","ntpm":310.9},{"tissue":"liver","ntpm":401.8}],"url":"https://www.proteinatlas.org/search/BHMT2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9H2M3","domains":[{"cath_id":"3.20.20.330","chopping":"12-312","consensus_level":"high","plddt":97.0197,"start":12,"end":312},{"cath_id":"-","chopping":"320-347","consensus_level":"medium","plddt":97.4054,"start":320,"end":347}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H2M3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H2M3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H2M3-F1-predicted_aligned_error_v6.png","plddt_mean":95.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BHMT2","jax_strain_url":"https://www.jax.org/strain/search?query=BHMT2"},"sequence":{"accession":"Q9H2M3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H2M3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H2M3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H2M3"}},"corpus_meta":[{"pmid":"18230605","id":"PMC_18230605","title":"Betaine-homocysteine S-methyltransferase-2 is an S-methylmethionine-homocysteine methyltransferase.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18230605","citation_count":67,"is_preprint":false},{"pmid":"25856673","id":"PMC_25856673","title":"Genome-wide expression in visceral adipose tissue from obese prepubertal children.","date":"2015","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/25856673","citation_count":65,"is_preprint":false},{"pmid":"21254359","id":"PMC_21254359","title":"Folate pathway and nonsyndromic cleft lip and palate.","date":"2010","source":"Birth defects research. 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The Km for SMM is 0.94 mM, and turnover is similar to BHMT. Methionine is a stronger inhibitor of BHMT2 than BHMT; S-adenosylmethionine is a weak inhibitor of BHMT2 but does not inhibit BHMT; dimethylglycine and betaine do not inhibit BHMT2.\",\n      \"method\": \"In vitro enzymatic assay with purified recombinant protein; kinetic parameter determination; inhibitor profiling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution with purified recombinant enzyme, kinetic characterization, and substrate/inhibitor profiling in a single rigorous study\",\n      \"pmids\": [\"18230605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BHMT2 protein expressed in mammalian (COS-1) cells is unstable and rapidly degraded in rabbit reticulocyte lysate, but can be stabilized by co-transfection with BHMT; after co-transfection BHMT2 co-precipitates with BHMT, indicating a physical interaction between the two proteins.\",\n      \"method\": \"Co-transfection of COS-1 cells, co-immunoprecipitation, rabbit reticulocyte lysate degradation assay\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-immunoprecipitation and degradation assay from a single lab, two complementary methods\",\n      \"pmids\": [\"18457970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mouse Bhmt2, acting through its substrate S-methylmethionine (SMM), protects against acetaminophen-induced liver toxicity in vivo by influencing methionine and glutathione biosynthesis; this protective effect is diet-dependent because SMM is only synthesized in plants.\",\n      \"method\": \"Integrative genomic analysis across multiple inbred mouse strains combined with NMR-based metabolomics; in vivo dietary manipulation\",\n      \"journal\": \"Genome research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multi-strain genetic analysis combined with metabolomics and in vivo dietary intervention, replicated across mouse strains\",\n      \"pmids\": [\"19923254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The human BHMT2 gene encodes a predicted 363-amino-acid protein (40.3 kDa) with 73% amino acid identity to BHMT. BHMT2 mRNA is most abundant in adult liver and kidney, with reduced expression in brain, heart, and skeletal muscle.\",\n      \"method\": \"cDNA cloning, gene sequencing, physical mapping, Northern blot/expression analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — cDNA cloning and expression profiling from a single lab, multiple tissues examined\",\n      \"pmids\": [\"11087663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mouse liver extracts containing BHMT2 recognize (S,S)-AdoMet but NOT (R,S)-AdoMet, and no enzymatic breakdown of (R,S)-AdoMet was detected in these extracts, indicating BHMT2 does not metabolize the age-damaged AdoMet diastereomer.\",\n      \"method\": \"In vitro enzyme assay with mouse liver extracts; substrate specificity testing\",\n      \"journal\": \"Rejuvenation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct in vitro assay with native liver extracts, single lab, negative result regarding (R,S)-AdoMet as substrate\",\n      \"pmids\": [\"20370499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In Bhmt knockout mice, Bhmt2 expression is upregulated in the cochlea following noise damage, suggesting a compensatory role for BHMT2 in cochlear methionine metabolism when BHMT is absent.\",\n      \"method\": \"Bhmt knockout mouse model; quantitative RT-PCR of cochlear gene expression; RNA arrays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function mouse model with transcriptional readout, single lab, compensatory upregulation inferred from gene expression data\",\n      \"pmids\": [\"30753104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPIC transcription factor binds to enhancer elements near Bhmt2 (and Bhmt, Dmgdh) in mouse embryonic stem cells and stabilizes NANOG binding, controlling betaine-dependent one-carbon metabolism including SMM-to-SAH flux and histone methylation marks (H3R17me2 and H3K4me3).\",\n      \"method\": \"ChIP-seq, gain-of-function and loss-of-function experiments in ESCs, metabolic flux analysis\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq binding data combined with gain/loss-of-function experiments and metabolic readout in a single lab study\",\n      \"pmids\": [\"37595034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In intestinal epithelial cells during necrotizing enterocolitis, BHMT2 and MAT1A regulate AHSG expression through S-adenosylmethionine production and histone methylation; silencing BHMT2 in LPS-stimulated HPIECs attenuates M1 macrophage polarization, inflammatory cytokine production, and macrophage invasive capacity in a transwell co-culture system, while BHMT2 overexpression promotes M1 macrophage activation.\",\n      \"method\": \"siRNA knockdown and overexpression in human primary intestinal epithelial cells; transwell co-culture with THP-1 macrophages; chromatin immunoprecipitation; RT-qPCR; Western blotting; ELISA; flow cytometry; NEC mouse model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (ChIP, co-culture, in vivo mouse model, gain/loss of function), single lab\",\n      \"pmids\": [\"41219226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Porcine BHMT2 protein (363 amino acids) shares 78% amino acid identity with porcine BHMT, and both genes have 8 exons spanning ~16 kb (BHMT2) and ~26 kb (BHMT). BHMT2 transcripts are most abundant in liver and kidney cortex in pigs, mirroring the human expression pattern; neither gene has a TATA or CAAT box but both have a CpG island at the promoter/transcriptional start site.\",\n      \"method\": \"cDNA cloning, RLM-RACE, gene structure analysis, qPCR expression profiling\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — molecular characterization in a pig model, single lab, primarily structural/expression data\",\n      \"pmids\": [\"21156199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Evolutionary analysis across 38 deuterostome species showed that BHMT2 arose by gene duplication from BHMT after divergence of mammals from other vertebrates, with accelerated evolutionary rates relative to BHMT; codons under positive selection map to enzymatic or oligomerization domains, suggesting involvement in enzyme function. Two deletions in BHMT2 relative to BHMT affect oligomerization and methyl donor specificity.\",\n      \"method\": \"Comparative genomics and evolutionary rate analysis (dN/dS, GA Branch analysis) across 38 species\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Moderate — computational/comparative genomic analysis, no direct biochemical experiment; replicated across many species but purely computational\",\n      \"pmids\": [\"26213999\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BHMT2 is a mammalian zinc metalloenzyme that remethylates homocysteine to methionine using S-methylmethionine (SMM) — not betaine — as the methyl donor; it is expressed primarily in liver and kidney, is stabilized by physical interaction with BHMT, protects against acetaminophen-induced hepatotoxicity in a diet-dependent manner through effects on methionine and glutathione biosynthesis, and in intestinal epithelial cells regulates M1 macrophage activation via an SAM/histone methylation-dependent axis involving MAT1A and AHSG.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BHMT2 is a mammalian zinc metalloenzyme of hepatic and renal one-carbon metabolism that remethylates homocysteine to methionine, using S-methylmethionine (SMM) rather than betaine as its methyl donor — a substrate specificity that distinguishes it from its paralog BHMT [#0, #3]. Kinetic characterization of purified recombinant enzyme established an SMM Km of ~0.94 mM with turnover comparable to BHMT, and a distinct inhibitor profile in which methionine is a strong inhibitor and S-adenosylmethionine a weak one, while betaine and dimethylglycine do not inhibit [#0]. The protein is intrinsically unstable in mammalian cells and is stabilized through direct physical interaction with BHMT, with which it co-precipitates [#1]. Through its SMM-dependent contribution to methionine and downstream glutathione biosynthesis, BHMT2 protects against acetaminophen-induced hepatotoxicity in a diet-dependent manner, since SMM is supplied only by plant sources [#2]. Beyond classical one-carbon metabolism, BHMT2 feeds S-adenosylmethionine production and histone methylation: acting with MAT1A in intestinal epithelial cells, it regulates AHSG expression and drives M1 macrophage activation and inflammatory cytokine output [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Before its biochemistry was known, the question was whether the BHMT-like gene encoded a distinct, tissue-restricted enzyme; cloning defined a 363-residue protein highly similar to BHMT but expressed predominantly in liver and kidney.\",\n      \"evidence\": \"cDNA cloning, physical mapping and Northern expression profiling of human BHMT2\",\n      \"pmids\": [\"11087663\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic activity or substrate demonstrated\", \"Subcellular localization not established\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"The central mechanistic question — what reaction BHMT2 catalyzes — was answered by showing it is a zinc metalloenzyme that remethylates homocysteine using SMM, not betaine, defining its functional divergence from BHMT.\",\n      \"evidence\": \"In vitro enzymatic assay with purified recombinant enzyme, kinetic and inhibitor profiling\",\n      \"pmids\": [\"18230605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal structure or catalytic-mechanism model\", \"Physiological flux through the SMM pathway in vivo not quantified in this study\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Why BHMT2 protein is hard to express was addressed by showing it is rapidly degraded but stabilized by BHMT, establishing a physical interaction with its paralog as a determinant of protein stability.\",\n      \"evidence\": \"Co-transfection in COS-1 cells, co-immunoprecipitation, reticulocyte lysate degradation assay\",\n      \"pmids\": [\"18457970\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, no reciprocal IP or stoichiometry of the BHMT/BHMT2 complex\", \"Degradation pathway and ubiquitin involvement unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The physiological consequence of BHMT2 activity was tied to hepatoprotection: through SMM it influences methionine and glutathione biosynthesis to limit acetaminophen toxicity, with the effect gated by dietary SMM availability.\",\n      \"evidence\": \"Multi-strain mouse genetic analysis with NMR metabolomics and in vivo dietary manipulation\",\n      \"pmids\": [\"19923254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic step in vivo inferred rather than measured\", \"Human dietary relevance not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Substrate-specificity boundaries were refined by testing AdoMet diastereomers, showing BHMT2-containing liver extracts handle (S,S)-AdoMet but do not metabolize the age-damaged (R,S)-AdoMet.\",\n      \"evidence\": \"In vitro assay with mouse liver extracts and diastereomer substrate testing\",\n      \"pmids\": [\"20370499\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Used crude extracts, not purified BHMT2\", \"Negative result does not pinpoint the responsible enzyme\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether BHMT2 can compensate for BHMT loss was addressed by showing its transcriptional upregulation in cochlea of Bhmt knockout mice after noise damage, implicating a backup role in tissue methionine metabolism.\",\n      \"evidence\": \"Bhmt knockout mouse, cochlear qRT-PCR and RNA arrays\",\n      \"pmids\": [\"30753104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Compensation inferred from mRNA only, not protein or activity\", \"Functional rescue not demonstrated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Transcriptional control of the SMM/one-carbon module was placed under SPIC, which binds Bhmt2 enhancers and stabilizes NANOG to govern SMM-to-SAH flux and histone methylation in embryonic stem cells.\",\n      \"evidence\": \"ChIP-seq with gain/loss-of-function and metabolic flux analysis in mouse ESCs\",\n      \"pmids\": [\"37595034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct contribution of BHMT2 versus co-regulated Bhmt/Dmgdh not separated\", \"Mechanism in differentiated tissues unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"An immunometabolic role was established: in intestinal epithelial cells BHMT2 with MAT1A controls AHSG via SAM and histone methylation, and BHMT2 levels bidirectionally set M1 macrophage polarization and inflammatory output.\",\n      \"evidence\": \"siRNA/overexpression in primary intestinal epithelial cells, transwell co-culture with macrophages, ChIP, and NEC mouse model\",\n      \"pmids\": [\"41219226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; causal chain from BHMT2 enzymatic activity to histone marks not fully reconstituted\", \"Direct enzymatic versus structural contribution of BHMT2 not dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how BHMT2 catalysis is structurally organized and how its SMM-derived methyl flux is mechanistically linked to the SAM/histone-methylation signaling roles emerging in stem cells and immune contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental structure of BHMT2\", \"No defined regulatory mechanism connecting metabolic activity to chromatin output\", \"Human in vivo physiological role beyond mouse models uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BHMT\", \"MAT1A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":5,"faith_pct":80.0}}