{"gene":"TG_312930","gene_symbol":"CGL","run_date":"2026-09-08T15:15:13","timeline":{"discoveries":[{"year":2018,"finding":"Recombinant TgCGL almost exclusively catalyzes α,γ-hydrolysis of l-cystathionine to form l-cysteine, with marginal reactivity toward l-cysteine; active-site residues Ser77 and Asn360 (vs. Glu59 and Ser340 in human CGL) are key determinants of substrate specificity — mutation Asn360Ser shifts specificity toward both α,β- and α,γ-elimination, while Ser77Glu completely abolishes activity toward l-cystathionine.","method":"Recombinant enzyme expression and biochemical characterization; structure-guided homology modelling; active-site mutagenesis (N360S and S77E variants) with kinetic assays","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis, single lab but multiple orthogonal methods (kinetics + structural modelling + mutagenesis)","pmids":["30036991"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"Crystal structures of native TgCGL and the variant TgCGL-N360S determined in complex with cystathionine, cysteine, and the inhibitor d,l-propargylglycine (PPG) reveal the binding mode of each molecule within the catalytic cavity; PPG acts as an irreversible inhibitor via a specific mechanism in the active site, and cysteine also inhibits the enzyme; N360S substitution at the active site broadens substrate specificity so that TgCGL can cleave both the CγS and CβS bonds of cystathionine.","method":"X-ray crystallography of native TgCGL and TgCGL-N360S co-crystallized with substrates/inhibitors; structural analysis of inhibitor binding modes","journal":"Protein science : a publication of the Protein Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures with bound ligands and mutagenesis, single lab with multiple orthogonal structural and biochemical methods","pmids":["36883335"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"TgCGL functions as the downstream enzyme in the reverse transsulfuration pathway of T. gondii, hydrolyzing cystathionine produced by TgCBS to yield cysteine; together, TgCBS and TgCGL constitute an intact, functional reverse transsulfuration pathway for cysteine biosynthesis in the parasite.","method":"Biochemical characterization of recombinant TgCBS and pathway reconstitution in vitro; demonstration that TgCBS produces cystathionine via PLP-catalyzed β-replacement using serine or O-acetylserine, which TgCGL then converts to cysteine","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of the two-step pathway with recombinant enzymes, replicated across multiple studies from same group using orthogonal methods","pmids":["32887901"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgCGL is a PLP-dependent enzyme that catalyzes the α,γ-elimination of l-cystathionine to produce l-cysteine, α-ketobutyrate, and ammonia as the terminal step of the reverse transsulfuration pathway in Toxoplasma gondii; active-site residues Ser77 and Asn360 (divergent from the human enzyme) determine its near-exclusive specificity for cystathionine over cysteine, the inhibitor d,l-propargylglycine binds irreversibly in the catalytic cavity, and structural studies have defined the molecular basis of substrate binding and inhibition."},"narrative":{"mechanistic_narrative":"TgCGL is a PLP-dependent enzyme that catalyzes the terminal step of the reverse transsulfuration pathway for cysteine biosynthesis in Toxoplasma gondii, acting downstream of TgCBS to hydrolyze cystathionine to cysteine [PMID:32887901]. Recombinant TgCGL almost exclusively performs α,γ-elimination of l-cystathionine, with only marginal reactivity toward l-cysteine, a near-exclusive substrate specificity dictated by active-site residues Ser77 and Asn360 that diverge from the human enzyme: mutation Asn360Ser broadens activity to include both α,β- and α,γ-elimination (cleavage of both the CγS and CβS bonds), while Ser77Glu abolishes activity toward cystathionine [PMID:30036991, PMID:36883335]. Crystal structures of native TgCGL and the TgCGL-N360S variant bound to cystathionine, cysteine, and the inhibitor d,l-propargylglycine define the molecular basis of substrate binding, with propargylglycine acting as an irreversible active-site inhibitor and cysteine also inhibiting the enzyme [PMID:36883335].","teleology":[{"year":2018,"claim":"Establishing what reaction TgCGL catalyzes and why its specificity differs from the human enzyme addressed whether the parasite enzyme is a selective drug target; the work showed it is a near-exclusive cystathionine α,γ-lyase governed by two divergent active-site residues.","evidence":"Recombinant enzyme kinetics with structure-guided homology modelling and active-site mutagenesis (N360S, S77E variants)","pmids":["30036991"],"confidence":"High","gaps":["Specificity determinants inferred from homology model rather than experimental structure at this stage","No in vivo demonstration of enzyme function in the parasite"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Whether TgCGL operates within a complete cysteine-biosynthesis route was unresolved; reconstituting the two-step pathway showed TgCBS and TgCGL together form an intact reverse transsulfuration pathway, placing TgCGL as the cystathionine-consuming terminal enzyme.","evidence":"In vitro reconstitution with recombinant TgCBS and TgCGL, demonstrating TgCBS-generated cystathionine is converted to cysteine by TgCGL","pmids":["32887901"],"confidence":"High","gaps":["Pathway shown in vitro; flux and essentiality in living parasites not established","Regulation of the pathway not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"The molecular basis of substrate binding and inhibition was undefined; crystal structures of native and N360S TgCGL with bound cystathionine, cysteine, and propargylglycine resolved the catalytic cavity, defined the irreversible inhibition mechanism, and confirmed that N360S broadens bond cleavage to both CγS and CβS.","evidence":"X-ray crystallography of native TgCGL and TgCGL-N360S co-crystallized with substrates and the inhibitor d,l-propargylglycine","pmids":["36883335"],"confidence":"High","gaps":["No structure-based inhibitor optimization or selectivity validation against human CGL reported","Cellular consequence of enzyme inhibition in the parasite not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"Whether TgCGL and the reverse transsulfuration pathway are essential for T. gondii viability or virulence in vivo, and whether active-site inhibitors translate into antiparasitic efficacy, remains uncharacterized in the available corpus.","evidence":"","pmids":[],"confidence":"Low","gaps":["No genetic knockout or in vivo essentiality data","No demonstration of antiparasitic activity for propargylglycine or other inhibitors"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGCBS (FUNCTIONAL PAIRING IN THE REVERSE TRANSSULFURATION PATHWAY; TGCBS PRODUCES CYSTATHIONINE THAT TGCGL CONVERTS TO CYSTEINE) [#2]"],"other_free_text":["Active-site residues Ser77 and Asn360 (vs. Glu59 and Ser340 in human CGL) determine near-exclusive cystathionine specificity; Ser77Glu abolishes activity, Asn360Ser broadens it to both α,β- and α,γ-elimination [#0, #1]","d,l-propargylglycine (PPG) is an irreversible active-site inhibitor; cysteine also inhibits the enzyme [#1]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"32887901","id":"PMC_32887901","title":"Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32887901","citation_count":26,"is_preprint":false},{"pmid":"30036991","id":"PMC_30036991","title":"Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii.","date":"2018","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/30036991","citation_count":12,"is_preprint":false},{"pmid":"34194677","id":"PMC_34194677","title":"Structural insight into the unique conformation of cystathionine β-synthase from Toxoplasma gondii.","date":"2021","source":"Computational and structural biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/34194677","citation_count":10,"is_preprint":false},{"pmid":"36883335","id":"PMC_36883335","title":"Structural basis of the inhibition of cystathionine γ-lyase from Toxoplasma gondii by propargylglycine and cysteine.","date":"2023","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/36883335","citation_count":9,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4542,"output_tokens":1103,"usd":0.015085,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6059,"output_tokens":2145,"usd":0.04196,"stage2_stop_reason":"end_turn"},"total_usd":0.057045,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"Recombinant TgCGL almost exclusively catalyzes α,γ-hydrolysis of l-cystathionine to form l-cysteine, with marginal reactivity toward l-cysteine; active-site residues Ser77 and Asn360 (vs. Glu59 and Ser340 in human CGL) are key determinants of substrate specificity — mutation Asn360Ser shifts specificity toward both α,β- and α,γ-elimination, while Ser77Glu completely abolishes activity toward l-cystathionine.\",\n      \"method\": \"Recombinant enzyme expression and biochemical characterization; structure-guided homology modelling; active-site mutagenesis (N360S and S77E variants) with kinetic assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with active-site mutagenesis, single lab but multiple orthogonal methods (kinetics + structural modelling + mutagenesis)\",\n      \"pmids\": [\"30036991\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Crystal structures of native TgCGL and the variant TgCGL-N360S determined in complex with cystathionine, cysteine, and the inhibitor d,l-propargylglycine (PPG) reveal the binding mode of each molecule within the catalytic cavity; PPG acts as an irreversible inhibitor via a specific mechanism in the active site, and cysteine also inhibits the enzyme; N360S substitution at the active site broadens substrate specificity so that TgCGL can cleave both the CγS and CβS bonds of cystathionine.\",\n      \"method\": \"X-ray crystallography of native TgCGL and TgCGL-N360S co-crystallized with substrates/inhibitors; structural analysis of inhibitor binding modes\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures with bound ligands and mutagenesis, single lab with multiple orthogonal structural and biochemical methods\",\n      \"pmids\": [\"36883335\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TgCGL functions as the downstream enzyme in the reverse transsulfuration pathway of T. gondii, hydrolyzing cystathionine produced by TgCBS to yield cysteine; together, TgCBS and TgCGL constitute an intact, functional reverse transsulfuration pathway for cysteine biosynthesis in the parasite.\",\n      \"method\": \"Biochemical characterization of recombinant TgCBS and pathway reconstitution in vitro; demonstration that TgCBS produces cystathionine via PLP-catalyzed β-replacement using serine or O-acetylserine, which TgCGL then converts to cysteine\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of the two-step pathway with recombinant enzymes, replicated across multiple studies from same group using orthogonal methods\",\n      \"pmids\": [\"32887901\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgCGL is a PLP-dependent enzyme that catalyzes the α,γ-elimination of l-cystathionine to produce l-cysteine, α-ketobutyrate, and ammonia as the terminal step of the reverse transsulfuration pathway in Toxoplasma gondii; active-site residues Ser77 and Asn360 (divergent from the human enzyme) determine its near-exclusive specificity for cystathionine over cysteine, the inhibitor d,l-propargylglycine binds irreversibly in the catalytic cavity, and structural studies have defined the molecular basis of substrate binding and inhibition.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgCGL is a PLP-dependent enzyme that catalyzes the terminal step of the reverse transsulfuration pathway for cysteine biosynthesis in Toxoplasma gondii, acting downstream of TgCBS to hydrolyze cystathionine to cysteine [#2]. Recombinant TgCGL almost exclusively performs α,γ-elimination of l-cystathionine, with only marginal reactivity toward l-cysteine, a near-exclusive substrate specificity dictated by active-site residues Ser77 and Asn360 that diverge from the human enzyme: mutation Asn360Ser broadens activity to include both α,β- and α,γ-elimination (cleavage of both the CγS and CβS bonds), while Ser77Glu abolishes activity toward cystathionine [#0, #1]. Crystal structures of native TgCGL and the TgCGL-N360S variant bound to cystathionine, cysteine, and the inhibitor d,l-propargylglycine define the molecular basis of substrate binding, with propargylglycine acting as an irreversible active-site inhibitor and cysteine also inhibiting the enzyme [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing what reaction TgCGL catalyzes and why its specificity differs from the human enzyme addressed whether the parasite enzyme is a selective drug target; the work showed it is a near-exclusive cystathionine α,γ-lyase governed by two divergent active-site residues.\",\n      \"evidence\": \"Recombinant enzyme kinetics with structure-guided homology modelling and active-site mutagenesis (N360S, S77E variants)\",\n      \"pmids\": [\"30036991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Specificity determinants inferred from homology model rather than experimental structure at this stage\",\n        \"No in vivo demonstration of enzyme function in the parasite\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Whether TgCGL operates within a complete cysteine-biosynthesis route was unresolved; reconstituting the two-step pathway showed TgCBS and TgCGL together form an intact reverse transsulfuration pathway, placing TgCGL as the cystathionine-consuming terminal enzyme.\",\n      \"evidence\": \"In vitro reconstitution with recombinant TgCBS and TgCGL, demonstrating TgCBS-generated cystathionine is converted to cysteine by TgCGL\",\n      \"pmids\": [\"32887901\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Pathway shown in vitro; flux and essentiality in living parasites not established\",\n        \"Regulation of the pathway not addressed\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The molecular basis of substrate binding and inhibition was undefined; crystal structures of native and N360S TgCGL with bound cystathionine, cysteine, and propargylglycine resolved the catalytic cavity, defined the irreversible inhibition mechanism, and confirmed that N360S broadens bond cleavage to both CγS and CβS.\",\n      \"evidence\": \"X-ray crystallography of native TgCGL and TgCGL-N360S co-crystallized with substrates and the inhibitor d,l-propargylglycine\",\n      \"pmids\": [\"36883335\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structure-based inhibitor optimization or selectivity validation against human CGL reported\",\n        \"Cellular consequence of enzyme inhibition in the parasite not tested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether TgCGL and the reverse transsulfuration pathway are essential for T. gondii viability or virulence in vivo, and whether active-site inhibitors translate into antiparasitic efficacy, remains uncharacterized in the available corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No genetic knockout or in vivo essentiality data\",\n        \"No demonstration of antiparasitic activity for propargylglycine or other inhibitors\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004123\", \"supporting_discovery_ids\": [0, 1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0030170\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0019344\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0019346\", \"supporting_discovery_ids\": [2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TgCBS (functional pairing in the reverse transsulfuration pathway; TgCBS produces cystathionine that TgCGL converts to cysteine) [#2]\"\n    ],\n    \"other_free_text\": [\n      \"Active-site residues Ser77 and Asn360 (vs. Glu59 and Ser340 in human CGL) determine near-exclusive cystathionine specificity; Ser77Glu abolishes activity, Asn360Ser broadens it to both α,β- and α,γ-elimination [#0, #1]\",\n      \"d,l-propargylglycine (PPG) is an irreversible active-site inhibitor; cysteine also inhibits the enzyme [#1]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_312930","name":"CGL","product":"cystathione gamma lyase","uniprot_acc":"A0A125YN40","chromosome":"TGME49_chrXI","start_pos":2986742},{"strain":"GT1","gene_id":"TGGT1_312930","name":null,"product":"putative cystathione gamma lyase","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":2949920},{"strain":"RH88","gene_id":"TGRH88_052040","name":null,"product":"putative cystathione gamma lyase","uniprot_acc":null,"chromosome":"CM023092","start_pos":3035623},{"strain":"VEG","gene_id":"TGVEG_312930","name":null,"product":"putative cystathione gamma lyase","uniprot_acc":"B6K8Y1","chromosome":"TGVEG_chrXI","start_pos":2999422}]}