PNPO is pyridoxine-5'-phosphate oxidase (pyridoxine/pyridoxamine 5'-phosphate oxidase; EC 1.4.3.5), an FMN-dependent flavoenzyme that catalyzes the terminal, rate-limiting step in the biosynthesis of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6. It oxidizes either pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to PLP, using molecular oxygen and generating hydrogen peroxide. The enzyme binds one FMN cofactor per subunit and functions as a homodimer, with each subunit binding one molecule of the product PLP tightly, giving product inhibition. PNPO acts in the cytosol and is broadly (housekeeping) expressed, with highest levels in liver, skeletal muscle and kidney. Because PLP is the essential cofactor for dozens of enzymes, including those of neurotransmitter metabolism, PNPO is critical for maintaining PLP supply; loss-of-function variants cause pyridoxal-5'-phosphate-responsive neonatal/early-infantile epileptic encephalopathy (PNPO deficiency).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004733 pyridoxamine phosphate oxidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Core catalytic molecular function of PNPO. The IBA propagation across the pyridoxamine 5'-phosphate oxidase family is correct and matches the experimentally established human activity (EC 1.4.3.5). The GO term definition explicitly covers oxidation of both pyridoxamine 5'-phosphate and pyridoxine 5'-phosphate to PLP. Supporting Evidence: file:human/PNPO/PNPO-uniprot.txt Catalyzes the oxidation of either pyridoxine 5'-phosphate |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Mitochondrial localization is not supported for human PNPO, which is a soluble cytosolic enzyme (Reactome places both reactions in the cytosol). This IBA call is driven by a subset of family members with divergent localization and represents an over-annotation for the human protein. Not removed (IBA can reflect genuine biology in some orthologs), but flagged. Propagation Review Root cause: PROPAGATION BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH |
| GO:0042823 pyridoxal 5'-phosphate biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Correct core biological process. PNPO catalyzes the terminal step of PLP (active vitamin B6) formation. Consistent with the UniProt PLP-salvage pathway annotation and with the experimental IDA annotation of the same term. Supporting Evidence: file:human/PNPO/PNPO-uniprot.txt 5'-phosphate from pyridoxamine 5'-phosphate: step 1/1. |
| GO:0004733 pyridoxamine phosphate oxidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (ARBA/InterPro/EC/RHEA) assignment of the correct core catalytic activity. Redundant with the experimental IDA/EXP annotations of the same term, which are the stronger evidence. Supporting Evidence: file:human/PNPO/PNPO-uniprot.txt Catalyzes the oxidation of either pyridoxine 5'-phosphate |
| GO:0008615 pyridoxine biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: This InterPro2GO/keyword-derived term mislabels the process. PNPO does not synthesize pyridoxine (vitamin B6); it oxidizes the already-phosphorylated B6 vitamers PNP and PMP to the active cofactor PLP. The specific, correct process term for this reaction is pyridoxal 5'-phosphate biosynthetic process. Suggest replacing with GO:0042823. Proposed replacements: pyridoxal 5'-phosphate biosynthetic process Supporting Evidence: file:human/PNPO/PNPO-uniprot.txt Catalyzes the oxidation of either pyridoxine 5'-phosphate |
| GO:0010181 FMN binding | IEA GO_REF:0000002 | ACCEPT | Summary: Correct and core. PNPO is a flavoprotein that binds one FMN per subunit as its redox cofactor. Supported experimentally (IDA, PMID:12824491) and by the crystal structure. Redundant with the IDA annotation of the same term. Supporting Evidence: file:human/PNPO/PNPO-uniprot.txt Binds 1 FMN per subunit. |
| GO:0016638 oxidoreductase activity, acting on the CH-NH2 group of donors | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Correct but overly general parent-type oxidoreductase term (relevant to the pyridoxamine-phosphate half-reaction, which acts on the CH-NH2 group of PMP). The specific child term GO:0004733 (pyridoxamine phosphate oxidase activity) is already annotated with experimental evidence and fully captures the activity, so this general term is redundant/over-general. |
| GO:0005515 protein binding | IPI PMID:25910212 Widespread macromolecular interaction perturbations in human... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a proteome-scale interactome screen (partner AGTRAP, Q6RW13-2). Non-informative for PNPO function - it identifies no specific molecular activity or biologically meaningful complex for this metabolic enzyme. Retained per policy (IPI not removed) but flagged as an over-annotation. |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a high-throughput interactome map (partner MTERF1, Q99551). Uninformative for the enzyme's function; kept but marked over-annotated. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a binary interactome reference map (partners AGTRAP Q6RW13-2 and LIME1 Q9H400). Uninformative; kept but marked over-annotated per curation policy on GO:0005515 IPI. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a proteome-scale interaction network (partner MTERF1, Q99551). Uninformative for PNPO function; kept but marked over-annotated. |
| GO:0004733 pyridoxamine phosphate oxidase activity | EXP PMID:15182361 Genomic organization, tissue distribution and deletion mutat... | ACCEPT | Summary: Direct experimental support for the core catalytic activity. Human brain PNPO was expressed in E. coli and its pyridox(am)ine 5'-phosphate oxidase activity assayed, including via N-/C-terminal truncation mutants. Core function. Supporting Evidence: PMID:15182361 affects neither the binding of coenzyme nor catalytic activity. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | MARK AS OVER ANNOTATED | Summary: Mitochondrial location from a high-throughput proteome dataset. PNPO is a canonical soluble cytosolic enzyme (Reactome localizes both catalyzed reactions to the cytosol), and the cited high-throughput mitochondrial proteome study does not specifically establish PNPO as a mitochondrial resident. Likely reflects cytosolic contamination of the mitochondrial preparation. Retained (HTP experimental-derived) but flagged as an over-annotation. |
| GO:0004733 pyridoxamine phosphate oxidase activity | IDA PMID:15772097 Neonatal epileptic encephalopathy caused by mutations in the... | ACCEPT | Summary: Direct experimental support for the core activity. Human PNPO variants expressed in CHO cells; the disease variant R229W markedly reduced pyridox(am)ine phosphate oxidase activity while splice/stop mutations were null, directly demonstrating the enzyme's catalytic function. Core function. Supporting Evidence: PMID:15772097 the missense mutation (R229W) markedly reduced pyridox(am)ine phosphate oxidase activity. |
| GO:0004733 pyridoxamine phosphate oxidase activity | IDA PMID:12824491 Structure and properties of recombinant human pyridoxine 5'-... | ACCEPT | Summary: Direct experimental support for the core catalytic activity. Recombinant human PNPO was characterized kinetically (low turnover, low-micromolar KM for both PNP and PMP) and structurally. Core function. Supporting Evidence: PMID:12824491 K(m) values in the low micromolar range for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. |
| GO:0010181 FMN binding | IDA PMID:12824491 Structure and properties of recombinant human pyridoxine 5'-... | ACCEPT | Summary: Correct and core. The crystal structure and biochemistry establish that human PNPO tightly binds one FMN per subunit as its flavin cofactor. Supporting Evidence: PMID:12824491 the binding sites for the tightly bound FMN and substrate are highly conserved. |
| GO:0030170 pyridoxal phosphate binding | IDA PMID:12824491 Structure and properties of recombinant human pyridoxine 5'-... | KEEP AS NON CORE | Summary: Experimentally supported - each subunit binds one molecule of the product pyridoxal 5'-phosphate tightly (product inhibition). This is a genuine ligand-binding property but is a consequence of the catalytic mechanism (PLP is the reaction product), not an independent core function; kept as non-core supporting evidence. Supporting Evidence: PMID:12824491 binds one molecule of pyridoxal 5'-phosphate tightly on each subunit. |
| GO:0042803 protein homodimerization activity | IDA PMID:12824491 Structure and properties of recombinant human pyridoxine 5'-... | KEEP AS NON CORE | Summary: Experimentally supported - the purified human enzyme is a homodimer, the catalytically active quaternary form. A real structural property but supportive rather than the enzyme's core catalytic function. Supporting Evidence: PMID:12824491 The purified human enzyme is a homodimer |
| GO:0042823 pyridoxal 5'-phosphate biosynthetic process | IDA PMID:12824491 Structure and properties of recombinant human pyridoxine 5'-... | ACCEPT | Summary: Correct core biological process, experimentally supported. PNPO catalyzes the terminal step in PLP synthesis. Supporting Evidence: PMID:12824491 Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-965019 | ACCEPT | Summary: Correct subcellular localization. PNPO is a soluble cytosolic enzyme; Reactome localizes the PNP-to-PLP oxidation reaction to the cytosol. Core location. |
| GO:0005829 cytosol | TAS Reactome:R-HSA-965079 | ACCEPT | Summary: Correct subcellular localization (Reactome reaction for the PMP-to-PLP oxidation, cytosol). Consistent with the companion cytosol annotation. |
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