Peptidylglycine alpha-amidating monooxygenase (PAM) of the Japanese quail, a bifunctional type I membrane enzyme of the regulated secretory pathway that generates C-terminally alpha-amidated neuropeptides and peptide hormones from their glycine-extended precursors. Its luminal region holds two catalytic domains. The N-terminal PHM domain (peptidylglycine alpha-hydroxylating monooxygenase) is a copper type II ascorbate-dependent monooxygenase: it binds two copper ions and uses molecular oxygen and ascorbate to hydroxylate the alpha-carbon of the C-terminal glycine. The PAL domain (peptidyl-alpha-hydroxyglycine alpha-amidating lyase) is a six-bladed NHL-repeat beta-propeller with a zinc active site; it cleaves the hydroxyglycine intermediate to release the amidated peptide and glyoxylate. An N-terminal signal peptide directs the protein into the secretory pathway, and a single transmembrane helix plus a disordered C-terminal cytosolic tail anchor it in the membranes of the trans-Golgi network and secretory granules. The tail carries the trafficking information that returns the protein through the endocytic pathway after exocytosis. This gene model lacks the linker between PHM and PAL that mammalian PAM-1 carries (exon 16), giving it the domain arrangement of the mammalian PAM-2 splice form, in which the two catalytic domains are rarely separated by cleavage. Soluble PHM and PAL, which arise from endoproteolysis of PAM, are released with the peptide products during regulated secretion.
Summary: Peptide amidation is the defining process of PAM, which performs both chemical steps of the conversion of a peptidylglycine to an amidated peptide.
Reason: The quail protein belongs to PANTHER subfamily PTHR10680:SF14, the same subfamily as the experimentally characterized human (P19021), rat (P14925), mouse and bovine PAM. In mammals PAM is the only known peptide-amidating enzyme. Both catalytic cores of the quail model are intact, and all PHM copper ligands and PAL zinc ligands are conserved (file:COTJA/A0A8C2TBA7/A0A8C2TBA7-bioinformatics/RESULTS-rereview.md). The gene product does the catalytic work of the process, so the participation test is met. The term is ARBA-derived, but the claim is independently supported.
Summary: The term is correct but uninformative. PAM's two specific activities are known and are already annotated.
Reason: The InterPro PHM/PAL signature (IPR000720) correctly identifies PAM as an enzyme, but catalytic activity is a root-level term. Following the refinement rule, the replacements are the two specific descendant activities that the gene already carries, GO:0004504 (PHM step) and GO:0004598 (PAL step). Both are listed because the bifunctional protein has both.
peptidylglycine Ξ±-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent Ξ±-hydroxylation of the peptidylglycine substrate; peptidyl-Ξ±-hydroxyglycine Ξ±-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate
Summary: The term is correct for the PHM domain but is a general ancestor of peptidylglycine monooxygenase activity.
Reason: The PHM domain (Cu2_ascorb_mOase N- and C-terminal domains, IPR000323 and IPR024548) is a copper monooxygenase. Its substrate is the C-terminal glycine of peptidylglycines, and the corresponding specific term, GO:0004504, is already on this gene. The previous review kept this row as non-core while arguing that it was too general; the action is now MODIFY to match that reason.
The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states.
Summary: This is the core activity of the PHM domain, the first step of peptide amidation.
Reason: The annotation combines an EC mapping with PANTHER placement. Its phylogenetic grounding is the PAINT IBD for GO:0004504 at PTN000073733 (Deuterostomia), whose seeds are human PAM (P19021), bovine PAM (P10731), mouse, rat and zebrafish PAM. Quail is a deuterostome and shares the SF14 subfamily with these seeds. Rat PHM structures place the substrate hydroxylation at a pair of copper sites. Both of those sites, with all six histidine and methionine ligands, are conserved in the quail model.
The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states.
The N-terminal PHM domain catalyzes the stereospecific hydroxylation of the Ξ±-carbon of the C-terminal glycine residue. This rate-limiting step requires three essential cofactors: (1) copper ions (two copper atoms per PHM domain), (2) molecular oxygen, and (3) reduced ascorbate as an electron donor
Summary: This is the core activity of the PAL domain, the second and final step of peptide amidation.
Reason: The annotation comes from an EC 4.3.2.5 mapping. It is supported because the quail protein has a complete NHL-repeat PAL domain (four NHL repeats, CDD NHL_PAL_like) with conserved zinc ligands, and because the structure of the PAL catalytic core of rat PAM, an SF14 subfamily member, establishes a Zn(II)-histidine active site. PAINT has no separate IBD for GO:0004598 in this family, so the support rests on the domain and residue conservation and on the shared subfamily with characterized mammalian PAM, not on an IBA.
The enzyme peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate.
Summary: The PHM domain binds two catalytic Cu(2+) ions. Copper binding is correct but is a cofactor property of the monooxygenase, not a separate function.
Reason: Copper binding is real and required. PIRSR places six catalytic copper ligands (H103, H104, H168, H238, H240, M310), and all of them are conserved relative to human PAM. The term is as precise as GO allows for a metal ligand. It describes the cofactor of the PHM activity, not the enzyme's role, so it is kept as non-core rather than accepted as a core function.
Summary: Soluble PHM and PAL derived from PAM are released with peptide products during regulated secretion and are found in serum and cerebrospinal fluid. This is a secondary location.
Reason: The TreeGrafter annotation comes from PTN000840922. The family PAINT table also has an IBD for GO:0005576 at PTN000840899 (taxon 6072, Eumetazoa), seeded by fly and rat genes. Release of soluble PHM and PAL is documented in mammals. This gene model lacks the exon-16 linker, so PAM-1-style separation of PHM from PAL is unlikely. Soluble bifunctional or PHM-containing products can still arise from cleavage near the membrane, and the term applies to the gene product rather than to one splice form. The protein amidates peptides in granules, not outside the cell, so the location is kept as non-core. The previous review accepted this term while calling it a secondary location; the action now matches that reasoning.
Both in neurons and endocrine cells, soluble PHM and PAL are secreted along with neuropeptides and peptide hormones upon stimulation; PHM and PAL can be detected in serum and cerebrospinal fluid
Summary: The term is correct but general. Its is_a descendant, peptide amidation (GO:0001519), is already on this gene and states the actual process.
Reason: GO:0001519 peptide amidation is_a GO:0006518 peptide metabolic process. Under the refinement rule, a correct but general term is MODIFY with the specific descendant, not KEEP_AS_NON_CORE as in the previous review.
Summary: PAM is a type I single-pass membrane protein. The specific membrane is the secretory granule membrane, a descendant of membrane.
Reason: The quail model has a SignalP signal peptide (1-21) and a Phobius transmembrane helix (734-758), and the juxtamembrane, transmembrane and cytosolic segments of mammalian PAM are all present. Mammalian PAM is a granule membrane protein whose cytosolic domain directs it to the trans-Golgi network and secretory granules. GO:0030667 secretory granule membrane is_a (via vesicle membrane) GO:0016020.
an epitope-tagged protein consisting of the transmembrane and cytosolic domains of PAM localizes to the trans Golgi network and secretory granules
GO:0016715 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced ascorbate as one donor, and incorporation of one atom of oxygen
IEA GO_REF:0000002
MODIFY
Summary: The term correctly describes the ascorbate-dependent PHM chemistry and is an ancestor of peptidylglycine monooxygenase activity.
Reason: GO:0004504 peptidylglycine monooxygenase activity is_a GO:0016715, and the gene already carries GO:0004504. The previous review kept this row as non-core while arguing that it was too general; the action now matches the reason.
Summary: The term comes from UniProt's "secretory vesicle membrane" subcellular location, but the vesicles PAM resides in are dense-core secretory granules, for which GO has the more accurate term secretory granule membrane.
Reason: The UniProt subcellular-location statement on this unreviewed entry is ARBA-derived. It maps through GO_REF:0000044 to transport vesicle membrane. The independent evidence places membrane PAM in regulated secretory granules and the trans-Golgi network, from which it is retrieved through the endocytic pathway after exocytosis. GO:0030667 secretory granule membrane is the better-supported term. It is not an is_a descendant of GO:0030658 in the current ontology, so this is a lateral correction to the term that the core function already uses.
Unlike soluble granule content proteins, granule membrane proteins are not secreted; after exocytosis and insertion into the plasma membrane, membrane PAM can be retrieved, traveling through the endocytic pathway in a regulated manner.
PAM is packaged into regulated secretory granules and neurosecretory vesicles, where it exists in both membrane-associated and soluble forms. In atrial cardiomyocytes, PAM represents a major membrane protein of secretory granules
Core Functions
The PHM domain catalyzes the first step of peptide C-terminal amidation, the copper- and ascorbate-dependent alpha-hydroxylation of the C-terminal glycine of peptidylglycine hormone and neuropeptide precursors in the lumen of the secretory pathway.
The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states.
All six PHM copper ligands are conserved (human H102, H103, H167, H237, H239, M309 -> quail H103, H104, H168, H238, H240, M310).
The PAL domain catalyzes the second step of peptide C-terminal amidation. Its zinc-dependent active site cleaves the peptidyl-alpha-hydroxyglycine intermediate made by PHM, releasing the alpha-amidated peptide and glyoxylate.
Q: Which PAM splice forms are expressed in quail neuroendocrine tissues, and is the exon-16-skipped (PAM-2-like) model A0A8C2TBA7 a major transcript?
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
Japanese quail PAM (PANTHER PTHR10680:SF14) is a bifunctional peptidylglycine alpha-amidating monooxygenase with an ascorbate-dependent dicopper PHM module and a zinc-dependent PAL beta-propeller module. The peptidylamidoglycolate lyase prediction matches an annotation the gene already carries and that its gene review accepts. The L-ascorbic acid binding prediction is absent from the gene's annotations and is supported by kinetic evidence of saturable ascorbate binding to rat PHM, a member of the same PANTHER subfamily.
Prediction method: ProtNLM2 Β· Version: UniProt 2024_06 pilot
Review rationale: The quail protein is in PANTHER subfamily PTHR10680:SF14 with experimentally characterized mammalian PAM and carries a complete NHL-repeat PAL domain. The crystal structure and mutagenesis of the rat PAL catalytic core establish N-dealkylation of the peptidyl-alpha-hydroxyglycine to the amidated peptide and glyoxylate (PMID:19604476), which justifies transfer of this activity within the subfamily. The gene already carries this exact term (GO:0004598, from EC 4.3.2.5 mapping), and its gene review accepts it as a core function, so the prediction is correct but not novel.
PMID:19604476: "alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-amidated peptide and glyoxylate."
Prediction method: ProtNLM2 Β· Version: UniProt 2024_06 pilot
Review rationale: Ascorbate is the reductant co-substrate of the PHM module, which carries the copper type II ascorbate-dependent monooxygenase domains (IPR000323, IPR014784). Stopped-flow reduction kinetics on rat PHM, from the same PANTHER subfamily (SF14), show saturable ascorbate dependence (KD about 1.1 mM), indicating that ascorbate binds the enzyme to reduce the copper centres (PMID:35380039). The same study notes that crystallography has not identified a discrete ascorbate site, so the binding is kinetically defined and of low affinity. The gene's annotations include the ascorbate-dependent monooxygenase activities (GO:0016715, GO:0004504) but no ascorbate binding term, and the PTHR10680 family review assigns L-ascorbic acid binding to PHM-bearing members. The prediction is therefore a supported, novel molecular function for this protein, though one that adds little beyond the co-substrate role implicit in its catalytic activity.
PMID:35380039: "The reduction obeys saturation kinetics with KD = 1120 ΞΌM and kobs = 146 sβ1 and indicates that ascorbate binds to the enzyme to accomplish reduction."
PMID:35380039: "crystallography failed to identify any"