{"id":"0d9fd8f5-372d-53e1-bc1d-d304848ae5b4","stable_key":"cb568d28-484a-5c2e-9fcc-2d780358e514:vc-enzyme-pam-two-domains","predicate":"contains-functional-domain","statement":"Expression of the bovine PHM-only construct in HEK293 cells increased secreted PHM activity, demonstrating a functional hydroxylating domain separable from the PAL portion of PAM.","claim_class":"mechanistic","status":"source_derived_draft","evidence_grade":"ungraded","direction":"positive","is_public":true,"mechanism_event_id":"e4b309a4-90ca-556e-a4e3-b3c5ec09ddeb","mechanism_event_label":"Peptide amidation begins with a separate vitamin-C-dependent hydroxylation step.","subject":{"id":"a34aa332-22a3-505c-899f-0e8a2b7bc2b3","slug":"bovine-pam","display_name":"Bovine peptidylglycine alpha-amidating monooxygenase","entity_type_key":"protein"},"object":{"id":"8e693d8d-d89a-5cb2-8fe9-9be1a4232cb0","slug":"bovine-phm-domain","display_name":"Bovine PAM peptidylglycine alpha-hydroxylating monooxygenase domain","entity_type_key":"protein_state"},"evidence_count":1,"mechanism_event":{"id":"e4b309a4-90ca-556e-a4e3-b3c5ec09ddeb","stable_key":"cb568d28-484a-5c2e-9fcc-2d780358e514:vc-enzyme-pam-two-domains-event","event_type":"biochemical_relationship","label":"Peptide amidation begins with a separate vitamin-C-dependent hydroxylation step.","description":"Expression of the bovine PHM-only construct in HEK293 cells increased secreted PHM activity, demonstrating a functional hydroxylating domain separable from the PAL portion of PAM.","status":"provisional","compartment":null,"participants":[{"entity":{"id":"a34aa332-22a3-505c-899f-0e8a2b7bc2b3","slug":"bovine-pam","display_name":"Bovine peptidylglycine alpha-amidating monooxygenase","entity_type_key":"protein"},"role":"parent-protein","stoichiometry":null,"state_label":"","sequence_order":0,"notes":""},{"entity":{"id":"8e693d8d-d89a-5cb2-8fe9-9be1a4232cb0","slug":"bovine-phm-domain","display_name":"Bovine PAM peptidylglycine alpha-hydroxylating monooxygenase domain","entity_type_key":"protein_state"},"role":"expressed-domain","stoichiometry":null,"state_label":"","sequence_order":1,"notes":""},{"entity":{"id":"38d0c4d9-53d1-5239-bfe3-c1b5e9b79085","slug":"ascorbate","display_name":"L-Ascorbate","entity_type_key":"small_molecule"},"role":"PHM-cofactor","stoichiometry":null,"state_label":"","sequence_order":2,"notes":""},{"entity":{"id":"2a870bb5-05a0-5e51-a70c-a9a843a8b571","slug":"copper-ii","display_name":"Copper(II) ion","entity_type_key":"ion"},"role":"PHM-metal-cofactor","stoichiometry":null,"state_label":"","sequence_order":3,"notes":""},{"entity":{"id":"899c7ab0-6f81-5b38-a6bd-fbb33d66ac8d","slug":"oxygen","display_name":"Molecular oxygen","entity_type_key":"small_molecule"},"role":"cosubstrate","stoichiometry":null,"state_label":"","sequence_order":4,"notes":""}]},"contexts":[{"dimension":"cross_nutrient","value_text":"Vitamin C chemistry in collagen, modified-lysine/carnitine metabolism or copper-dependent peptide/catecholamine processing.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"experimental_model","value_text":"Purified bovine pituitary PAL and bovine-domain expression constructs in HEK293 cells","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"exposure","value_text":"Purified PAL with N-acetyl-Tyr-Val-alpha-hydroxyglycine; PHM or PAL-containing constructs expressed in HEK293.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"limitations","value_text":"Bovine protein expressed in human cells is not a human PAM protein; domain construct activity does not quantify tissue hormone synthesis.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"nutrient_topic","value_text":"Vitamin C research collection; topical membership is not evidence of a direct dietary effect.","comparator":null,"unit":null,"notes":"","entity":{"slug":"vitamin-c","display_name":"Vitamin C","entity_type_key":"chemical_species"}},{"dimension":"organism","value_text":"Bos taurus (protein); Homo sapiens (expression host)","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"plain_language","value_text":"Peptide amidation begins with a separate vitamin-C-dependent hydroxylation step.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"primary_references","value_text":"[pal1991] Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. (1991). https://pubmed.ncbi.nlm.nih.gov/1902227/ DOI: 10.1016/s0021-9258(20)89524-6","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"tissue_or_cell_type","value_text":"Bovine neurointermediate pituitary; heterologous HEK293","comparator":null,"unit":null,"notes":"","entity":null}],"evidence":[{"id":"af6ff7fd-35f9-5fe3-b551-979221cf6a70","evidence_kind":"source_excerpt","locator":"Lines 885-896","start_line":885,"end_line":896,"excerpt":"### vc-enzyme-pam-two-domains\nExpression of the bovine PHM-only construct in HEK293 cells increased secreted PHM activity, demonstrating a functional hydroxylating domain separable from the PAL portion of PAM.\nCondition category: normal\nnutrient_topic: Vitamin C research collection; topical membership is not evidence of a direct dietary effect.\nplain_language: Peptide amidation begins with a separate vitamin-C-dependent hydroxylation step.\norganism: Bos taurus (protein); Homo sapiens (expression host)\ntissue_or_cell_type: Bovine neurointermediate pituitary; heterologous HEK293\nexperimental_model: Purified bovine pituitary PAL and bovine-domain expression constructs in HEK293 cells\nlimitations: Bovine protein expressed in human cells is not a human PAM protein; domain construct activity does not quantify tissue hormone synthesis.\ncross_nutrient: Vitamin C chemistry in collagen, modified-lysine/carnitine metabolism or copper-dependent peptide/catecholamine processing.\nexposure: Purified PAL with N-acetyl-Tyr-Val-alpha-hydroxyglycine; PHM or PAL-containing constructs expressed in HEK293.\n[pal1991] Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. (1991). https://pubmed.ncbi.nlm.nih.gov/1902227/ DOI: 10.1016/s0021-9258(20)89524-6","model_system":"Purified bovine pituitary PAL and bovine-domain expression constructs in HEK293 cells","directness":"author_interpretation","verification_status":"source_derived_draft","notes":"Exact curation-document quotation, not publisher quotation. Study references: [pal1991] Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression. (1991). https://pubmed.ncbi.nlm.nih.gov/1902227/ DOI: 10.1016/s0021-9258(20)89524-6","relationship":"supports","weight":1.0,"link_notes":"","source":{"id":"fa811221-13bd-5c10-adc1-eaf097c7703c","stable_key":"import-cb568d28-484a-5c2e-9fcc-2d780358e514","title":"Vitamin C: mechanisms, deficiency and nutrient interactions (2026-09-17)","document_type":"imported_text","citation_label":"AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text.","file_path":"","sha256":"b7fd83f956abb81855f2ea23199ba14e465cd91f4a2ac560277ec21ddafd7bfc","revision_id":"6cde9bbb-d712-5746-82a6-85b144253efa","review_status":"unverified_draft","notes":""}}],"relations":[],"conflicts":[],"corrections":[],"research":null}