{"gene":"TG_256760","gene_symbol":"PYKI","run_date":"2026-09-08T12:58:25","timeline":{"discoveries":[{"year":2010,"finding":"Crystal structure of T. gondii PYK1 (TgPYK1) was determined in both open and closed B-domain conformations, revealing that the closed conformation is inactive without any bound substrate — the first such observation for a PK enzyme. B-domain movements are coupled to changes in buried surface area between adjacent monomers in the tetramer, linking the catalytic cycle to domain rearrangements. Glucose-6-phosphate was confirmed as an allosteric activator of TgPYK1, and an unusual ordered conformation in the allosteric binding domain was attributed to an apicomplexan-specific insertion that explains activation by a mono-phosphorylated sugar.","method":"X-ray crystallography; enzymatic activity characterization; allosteric activator assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with two conformations plus enzymatic activity characterization in a single rigorous study","pmids":["20856875"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"PYK1 localizes to the cytosol and is the primary source of pyruvate in T. gondii. Conditional depletion of PYK1 caused global alteration of carbon metabolism, amylopectin accumulation, reduced cellular ATP, and severe growth impairment, demonstrating that PYK1 is essential for maintaining pyruvate homeostasis. Attenuated growth was partially rescued by lactate or alanine supplementation, and lactate rescue required lactate dehydrogenase activity to convert lactate to pyruvate, placing PYK1 in the central pyruvate-supply node of carbon metabolism.","method":"Conditional genetic depletion (knockdown); metabolic rescue experiments with lactate and alanine; lactate dehydrogenase inhibition; intracellular ATP measurement; amylopectin staining","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO with multiple orthogonal phenotypic readouts (metabolomics, ATP, amylopectin, growth) and specific rescue experiments in a single rigorous study","pmids":["31186321"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"PYK2 localizes to the apicoplast. Genetic deletion of PYK2 alone did not noticeably affect parasite growth or virulence, but combined depletion of PYK1 and ablation of PYK2 led to accentuated loss of apicoplasts and complete growth arrest, indicating cooperative pyruvate utilization between cytosol and apicoplast compartments.","method":"Genetic deletion of PYK2; conditional depletion of PYK1 combined with PYK2 deletion; apicoplast integrity assay; growth assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Moderate — double genetic manipulation with clear organelle and growth phenotypes, multiple orthogonal readouts in a single study","pmids":["31186321"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"T. gondii PYK1 is a cytosolic, tetrameric pyruvate kinase allosterically activated by glucose-6-phosphate (via an apicomplexan-specific domain) that is essential for pyruvate homeostasis, ATP production, and parasite growth, acting as the primary node where multiple carbon sources (glucose, lactate, alanine) converge on pyruvate, and cooperating with the apicoplast-localized PYK2 to maintain apicoplast integrity."},"narrative":{"mechanistic_narrative":"PYK1 is the cytosolic pyruvate kinase of Toxoplasma gondii and the central node at which multiple carbon sources converge to supply pyruvate for parasite metabolism [PMID:31186321]. Structurally, it is a tetramer whose catalytic cycle couples B-domain open/closed transitions to changes in the buried surface area between adjacent monomers; uniquely among pyruvate kinases, its fully closed conformation is inactive without bound substrate, and it is allosterically activated by glucose-6-phosphate through an ordered apicomplexan-specific insertion in the allosteric domain that accommodates the mono-phosphorylated sugar [PMID:20856875]. Conditional depletion of PYK1 globally reshapes carbon metabolism, causing amylopectin accumulation, reduced cellular ATP, and severe growth impairment, establishing PYK1 as essential for pyruvate homeostasis; growth is partially rescued by lactate or alanine, with lactate rescue requiring lactate dehydrogenase to regenerate pyruvate [PMID:31186321]. PYK1 acts cooperatively with the apicoplast-localized paralog PYK2: loss of PYK2 alone is tolerated, but combined loss of PYK1 and PYK2 accentuates apicoplast loss and arrests growth, indicating that cytosolic and apicoplast pyruvate pools are functionally linked [PMID:31186321].","teleology":[{"year":2010,"claim":"Establishing how a pyruvate kinase in an apicomplexan parasite is regulated required resolving its catalytic conformations and its unusual allosteric activator; the structure revealed a domain-coupled, glucose-6-phosphate-responsive enzyme distinct from canonical PKs.","evidence":"X-ray crystallography of TgPYK1 in open and closed B-domain states with enzymatic activity and allosteric activator assays","pmids":["20856875"],"confidence":"High","gaps":["Structure does not address the in vivo metabolic role or essentiality of PYK1","The physiological trigger driving glucose-6-phosphate activation in the intracellular parasite was not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Whether PYK1 is metabolically essential and how it integrates carbon flux was unknown; conditional depletion showed it is the primary pyruvate source and required for pyruvate homeostasis, ATP levels, and growth.","evidence":"Conditional knockdown with metabolomics, ATP measurement, amylopectin staining, and lactate/alanine rescue coupled to lactate dehydrogenase inhibition","pmids":["31186321"],"confidence":"High","gaps":["Does not resolve how flux is partitioned among glucose, lactate, and alanine inputs in vivo","The regulatory link between glucose-6-phosphate activation and the observed metabolic phenotype was not directly tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"The functional relationship between cytosolic PYK1 and the apicoplast paralog PYK2 was unclear; genetic combination showed the two cooperate to maintain apicoplast integrity.","evidence":"Genetic deletion of PYK2 with and without conditional PYK1 depletion, assayed for apicoplast integrity and growth","pmids":["31186321"],"confidence":"High","gaps":["The mechanism by which pyruvate pools are shared or shuttled between cytosol and apicoplast is not defined","Why PYK2 loss alone is dispensable but becomes critical upon PYK1 depletion is unexplained"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How glucose-6-phosphate allosteric activation is physiologically deployed to tune PYK1 flux during the intracellular lytic cycle, and the molecular basis of cytosol-apicoplast pyruvate cooperation, remain open.","evidence":"","pmids":[],"confidence":"High","gaps":["No study directly links the structural allosteric mechanism to in vivo metabolic regulation","No transporter or shuttle mediating cytosol-to-apicoplast pyruvate supply has been identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Homotetramer (PYK1)"],"partners":["PYK2 (FUNCTIONAL COOPERATION, APICOPLAST-LOCALIZED PARALOG)","LACTATE DEHYDROGENASE (REQUIRED FOR LACTATE-TO-PYRUVATE RESCUE)"],"other_free_text":["Allosterically activated by glucose-6-phosphate via an apicomplexan-specific insertion in the allosteric domain [#0]","Closed B-domain conformation is catalytically inactive without bound substrate, unusual among pyruvate kinases [#0]","Depletion causes amylopectin accumulation and reduced cellular ATP [#1]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"31186321","id":"PMC_31186321","title":"Pyruvate Homeostasis as a Determinant of Parasite Growth and Metabolic Plasticity in Toxoplasma gondii.","date":"2019","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/31186321","citation_count":61,"is_preprint":false},{"pmid":"20856875","id":"PMC_20856875","title":"The crystal structure of Toxoplasma gondii pyruvate kinase 1.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20856875","citation_count":22,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":3800,"output_tokens":1047,"usd":0.013553,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":5957,"output_tokens":2261,"usd":0.043155,"stage2_stop_reason":"end_turn"},"total_usd":0.056708,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of T. gondii PYK1 (TgPYK1) was determined in both open and closed B-domain conformations, revealing that the closed conformation is inactive without any bound substrate — the first such observation for a PK enzyme. B-domain movements are coupled to changes in buried surface area between adjacent monomers in the tetramer, linking the catalytic cycle to domain rearrangements. Glucose-6-phosphate was confirmed as an allosteric activator of TgPYK1, and an unusual ordered conformation in the allosteric binding domain was attributed to an apicomplexan-specific insertion that explains activation by a mono-phosphorylated sugar.\",\n      \"method\": \"X-ray crystallography; enzymatic activity characterization; allosteric activator assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with two conformations plus enzymatic activity characterization in a single rigorous study\",\n      \"pmids\": [\"20856875\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PYK1 localizes to the cytosol and is the primary source of pyruvate in T. gondii. Conditional depletion of PYK1 caused global alteration of carbon metabolism, amylopectin accumulation, reduced cellular ATP, and severe growth impairment, demonstrating that PYK1 is essential for maintaining pyruvate homeostasis. Attenuated growth was partially rescued by lactate or alanine supplementation, and lactate rescue required lactate dehydrogenase activity to convert lactate to pyruvate, placing PYK1 in the central pyruvate-supply node of carbon metabolism.\",\n      \"method\": \"Conditional genetic depletion (knockdown); metabolic rescue experiments with lactate and alanine; lactate dehydrogenase inhibition; intracellular ATP measurement; amylopectin staining\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with multiple orthogonal phenotypic readouts (metabolomics, ATP, amylopectin, growth) and specific rescue experiments in a single rigorous study\",\n      \"pmids\": [\"31186321\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PYK2 localizes to the apicoplast. Genetic deletion of PYK2 alone did not noticeably affect parasite growth or virulence, but combined depletion of PYK1 and ablation of PYK2 led to accentuated loss of apicoplasts and complete growth arrest, indicating cooperative pyruvate utilization between cytosol and apicoplast compartments.\",\n      \"method\": \"Genetic deletion of PYK2; conditional depletion of PYK1 combined with PYK2 deletion; apicoplast integrity assay; growth assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double genetic manipulation with clear organelle and growth phenotypes, multiple orthogonal readouts in a single study\",\n      \"pmids\": [\"31186321\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"T. gondii PYK1 is a cytosolic, tetrameric pyruvate kinase allosterically activated by glucose-6-phosphate (via an apicomplexan-specific domain) that is essential for pyruvate homeostasis, ATP production, and parasite growth, acting as the primary node where multiple carbon sources (glucose, lactate, alanine) converge on pyruvate, and cooperating with the apicoplast-localized PYK2 to maintain apicoplast integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PYK1 is the cytosolic pyruvate kinase of Toxoplasma gondii and the central node at which multiple carbon sources converge to supply pyruvate for parasite metabolism [#1]. Structurally, it is a tetramer whose catalytic cycle couples B-domain open/closed transitions to changes in the buried surface area between adjacent monomers; uniquely among pyruvate kinases, its fully closed conformation is inactive without bound substrate, and it is allosterically activated by glucose-6-phosphate through an ordered apicomplexan-specific insertion in the allosteric domain that accommodates the mono-phosphorylated sugar [#0]. Conditional depletion of PYK1 globally reshapes carbon metabolism, causing amylopectin accumulation, reduced cellular ATP, and severe growth impairment, establishing PYK1 as essential for pyruvate homeostasis; growth is partially rescued by lactate or alanine, with lactate rescue requiring lactate dehydrogenase to regenerate pyruvate [#1]. PYK1 acts cooperatively with the apicoplast-localized paralog PYK2: loss of PYK2 alone is tolerated, but combined loss of PYK1 and PYK2 accentuates apicoplast loss and arrests growth, indicating that cytosolic and apicoplast pyruvate pools are functionally linked [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing how a pyruvate kinase in an apicomplexan parasite is regulated required resolving its catalytic conformations and its unusual allosteric activator; the structure revealed a domain-coupled, glucose-6-phosphate-responsive enzyme distinct from canonical PKs.\",\n      \"evidence\": \"X-ray crystallography of TgPYK1 in open and closed B-domain states with enzymatic activity and allosteric activator assays\",\n      \"pmids\": [\"20856875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structure does not address the in vivo metabolic role or essentiality of PYK1\",\n        \"The physiological trigger driving glucose-6-phosphate activation in the intracellular parasite was not defined\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Whether PYK1 is metabolically essential and how it integrates carbon flux was unknown; conditional depletion showed it is the primary pyruvate source and required for pyruvate homeostasis, ATP levels, and growth.\",\n      \"evidence\": \"Conditional knockdown with metabolomics, ATP measurement, amylopectin staining, and lactate/alanine rescue coupled to lactate dehydrogenase inhibition\",\n      \"pmids\": [\"31186321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Does not resolve how flux is partitioned among glucose, lactate, and alanine inputs in vivo\",\n        \"The regulatory link between glucose-6-phosphate activation and the observed metabolic phenotype was not directly tested\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The functional relationship between cytosolic PYK1 and the apicoplast paralog PYK2 was unclear; genetic combination showed the two cooperate to maintain apicoplast integrity.\",\n      \"evidence\": \"Genetic deletion of PYK2 with and without conditional PYK1 depletion, assayed for apicoplast integrity and growth\",\n      \"pmids\": [\"31186321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The mechanism by which pyruvate pools are shared or shuttled between cytosol and apicoplast is not defined\",\n        \"Why PYK2 loss alone is dispensable but becomes critical upon PYK1 depletion is unexplained\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How glucose-6-phosphate allosteric activation is physiologically deployed to tune PYK1 flux during the intracellular lytic cycle, and the molecular basis of cytosol-apicoplast pyruvate cooperation, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No study directly links the structural allosteric mechanism to in vivo metabolic regulation\",\n        \"No transporter or shuttle mediating cytosol-to-apicoplast pyruvate supply has been identified\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004743\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0016208\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006096\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006090\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\"Homotetramer (PYK1)\"],\n    \"partners\": [\"PYK2 (functional cooperation, apicoplast-localized paralog)\", \"Lactate dehydrogenase (required for lactate-to-pyruvate rescue)\"],\n    \"other_free_text\": [\n      \"Allosterically activated by glucose-6-phosphate via an apicomplexan-specific insertion in the allosteric domain [#0]\",\n      \"Closed B-domain conformation is catalytically inactive without bound substrate, unusual among pyruvate kinases [#0]\",\n      \"Depletion causes amylopectin accumulation and reduced cellular ATP [#1]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":{"gene":"TG_256760","tier":"IDENTITY","verdict":"Identity concern","subtype":"paralog","uniprot_band":"empty","rules_fired":"R3","issue":"R3: narrative opens with 'PYK1 is' (query symbol is PYKI)"},"strains":[{"strain":"ME49","gene_id":"TGME49_256760","name":"PYKI","product":"pyruvate kinase PyK1","uniprot_acc":"A0A125YPV4","chromosome":"TGME49_chrVIIb","start_pos":4480660},{"strain":"GT1","gene_id":"TGGT1_256760","name":null,"product":"pyruvate kinase PyK1","uniprot_acc":null,"chromosome":"TGGT1_chrVIIb","start_pos":4413512},{"strain":"RH88","gene_id":"TGRH88_072970","name":null,"product":"pyruvate kinase PyK1","uniprot_acc":null,"chromosome":"CM023089","start_pos":4515061},{"strain":"VEG","gene_id":"TGVEG_256760","name":null,"product":"pyruvate kinase PyK1","uniprot_acc":"A0A0F7UWM4","chromosome":"TGVEG_chrVIIb","start_pos":4498479}]}