AMO (also called HPAO or HPAO-1) is the peroxisomal copper amine oxidase of the methylotrophic yeast Hansenula polymorpha (Ogataea angusta). It is a homodimeric quinoprotein of 692 residues in the copper amine oxidase family (PANTHER PTHR10638, subfamily SF86) that oxidatively deaminates small primary aliphatic amines, chiefly methylamine and ethylamine, using O2 to give the aldehyde, ammonia and hydrogen peroxide. Each subunit carries a type 2 copper ion held by three histidines (His456, His458, His624) and a topaquinone (TPQ) cofactor generated by copper- and oxygen-dependent self-oxidation of Tyr405; Asp319 is the catalytic base. The enzyme is induced when primary amines are the sole nitrogen source and is imported into the peroxisomal matrix through an N-terminal PTS2-type signal. There it catalyses the first step of methylamine use as a nitrogen source, releasing ammonia together with formaldehyde and H2O2, which peroxisomal catalase removes. A paralogous enzyme, HPAO-2, prefers aromatic amines. AMO is one of the most thoroughly studied copper amine oxidases structurally and mechanistically, from TPQ biogenesis to O2 access channels and substrate specificity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005507 copper ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: Copper binding is established directly for AMO. Crystal structures of the mature enzyme show one type 2 copper per subunit coordinated by His456, His458 and His624. Copper is required both for catalysis and for self-catalysed TPQ biogenesis. Reason: The electronic row agrees with experimental structural evidence on this protein [PMID:9551552; PMID:22691777]. A proteolytic C-terminal fragment that keeps copper and TOPA confirms that the metal is bound by the catalytic domain [PMID:7556609]. The family-root PAINT IBD for copper ion binding (PTN000067313) also covers SF86. Copper binding is a cofactor requirement of the core amine oxidase activity, so it is kept as a supporting part of that core function and not as a separate core activity. Supporting Evidence: PMID:9551552 CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. PMID:7556609 The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. PMID:22691777 TPQ is synthesized through the post-translational modification of an endogenous tyrosine residue and requires only oxygen and copper to proceed. file:PICAN/AMO/AMO-uniprot.txt CC Note=Binds 1 copper ion per subunit. |
| GO:0005777 peroxisome | IEA GO_REF:0000044 | MODIFY | Summary: AMO is a peroxisomal matrix enzyme in its native host. Cytochemistry places amine oxidase activity in peroxisomes of methylamine-grown cells, and targeting studies show that an N-terminal PTS2 signal imports AMO into the matrix. The more specific compartment is therefore supported. Reason: The annotation is correct but less specific than the evidence. Native-host experiments show that AMO is a peroxisomal matrix protein [PMID:8511963], that its N-terminal 16 residues direct a reporter to the peroxisomal matrix [PMID:7805876], and that its activity sits in catalase-containing microbodies [PMID:7192080]. Replace with GO:0005782 peroxisomal matrix, a part_of descendant of peroxisome. The C-terminal SRL (UniProt MOTIF 690..692, ECO:0000255) is not needed for targeting [PMID:8511963], so the localization rests on the PTS2 signal. The enzyme is cytosolic when expressed in S. cerevisiae [PMID:20155950]. That is a heterologous artefact and does not apply to the native host. Proposed replacements: peroxisomal matrix Supporting Evidence: PMID:8511963 Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. PMID:7805876 Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. PMID:7192080 Cytochemical staining experiments indicated that the amine oxidase activity was located in the microbodies in both organisms. |
| GO:0008131 primary methylamine oxidase activity | EXP PMID:7556609 Limited proteolysis of Hansenula polymorpha yeast amine oxid... | ACCEPT | Summary: AMO is a primary amine oxidase (EC 1.4.3.21) that converts methylamine and other small primary amines, with O2, to aldehyde, ammonia and H2O2. This paper characterises the cofactor-containing catalytic domain of the active recombinant enzyme. Reason: This is the core activity of AMO and has extensive experimental support from the protein of this species. UniProt credits this paper (ECO:0000269) for the catalytic activity. The cached abstract is limited to the cofactor-bearing proteolytic fragment, so the curator's EXP reading of the full text is not overruled. Independent papers confirm the activity: the native enzyme oxidises methylamine to ammonia, formaldehyde and H2O2 [PMID:7192080], the recombinant quinoprotein is as active as the native enzyme [PMID:8011631], and kinetic studies give kcat values for methylamine, ethylamine and benzylamine [PMID:17409383; PMID:20684524; PMID:23452079]. The family-root PAINT IBD for this term (PTN000067313) agrees. Supporting Evidence: PMID:7556609 The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase. PMID:7192080 The enzyme catalyses the oxidation of methylamine by molecular oxygen into ammonia, formaldehyde and hydrogen peroxide PMID:8011631 The recombinant enzyme is as active as the native enzyme in catalyzing methylamine oxidation. |
| GO:0008131 primary methylamine oxidase activity | EXP PMID:9551552 Copper amine oxidase from Hansenula polymorpha: the crystal ... | ACCEPT | Summary: The 2.4 A crystal structure shows the active TPQ conformation and the Asp319 base that together carry out primary amine oxidation by this methylamine oxidase. Reason: The structure, with the activity-abolishing D319N and activity-reducing D319E mutants [PMID:9551552; PMID:10387066], ties the catalytic mechanism of the methylamine oxidase reaction to this protein. It is consistent with the other EXP and IEA rows for the same term. Supporting Evidence: PMID:9551552 The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. PMID:9551552 These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. PMID:10387066 The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. |
| GO:0008131 primary methylamine oxidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The electronic annotation to primary methylamine oxidase activity agrees with the experimentally characterised activity of AMO. Reason: This row repeats the claim of the two EXP rows. Kinetic data on this protein support it directly: methylamine kcat 6.2 s-1 [PMID:17409383], ethylamine as the most efficient substrate [PMID:23452079], and a 330-fold preference for methylamine over benzylamine [PMID:20155950]. Supporting Evidence: file:PICAN/AMO/AMO-uniprot.txt kcat is 6.2 sec(-1) with methylamine as substrate (at pH 8 and PMID:23452079 Ethylamine is the most efficient known substrate for HPAO-1. PMID:20155950 In HPAO-1, the k(cat)/K(m) for methylamine is 330-fold |
| GO:0009308 amine metabolic process | IEA GO_REF:0000120 | MODIFY | Summary: AMO takes part in amine metabolism by catalysing its first, oxidative step: it degrades methylamine (and other small primary amines) to formaldehyde, ammonia and H2O2. For this enzyme the specific process is methylamine metabolism, a catabolic route that lets the cell use amines as a nitrogen source. Reason: The term is correct but general. The family-root PAINT IBD (PTN000067313) places it at this broad level for the whole heterogeneous family. For AMO, species-specific evidence supports the more specific descendants. AMO is the key enzyme of methylamine metabolism in H. polymorpha [PMID:7192080], is induced by primary amines as sole nitrogen source [PMID:8511963], and itself catalyses the deamination step, so it does the work of the process. Replace with GO:0030416 methylamine metabolic process (is_a amine metabolic process) and GO:0009310 amine catabolic process (is_a amine metabolic process). Proposed replacements: methylamine metabolic process amine catabolic process Supporting Evidence: PMID:7192080 it is considered to be the key enzyme in methylamine metabolism in the organisms studied PMID:8511963 which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source |
| GO:0048038 quinone binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: This InterPro2GO mapping refers to the topaquinone cofactor. In AMO the TPQ is Tyr405 of the polypeptide itself, oxidised in place, and not an exogenous quinone that the enzyme binds. Reason: Structures of the precursor and mature enzyme show that TPQ forms by copper- and O2-dependent self-oxidation of an endogenous tyrosine (Tyr405) [PMID:22691777; PMID:10933787; PMID:16245947]. No free quinone ligand is known to bind AMO. Quinone binding therefore describes a covalent cofactor residue as a binding activity and adds nothing beyond primary methylamine oxidase activity. GO has no current process term for topaquinone biogenesis (the peptidyl-topaquinone terms are obsolete). The row is kept flagged and not removed because the mapping reflects real quinone chemistry in the active site. Supporting Evidence: PMID:22691777 TPQ is synthesized through the post-translational modification of an endogenous tyrosine residue and requires only oxygen and copper to proceed. PMID:16245947 is generated from the post-translational modification of an active site tyrosine (Y405) in a reaction shown to be dependent on both molecular oxygen and a mononuclear copper center |
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Download this section (compressed HTML)Q: Does an amo deletion strain of H. polymorpha fail to grow on methylamine or ethylamine as sole nitrogen source, or can HPAO-2 compensate? No published deletion phenotype was found, so the role in nitrogen assimilation rests on induction, localization and enzymology.
Q: The UniProt MOTIF 690..692 "Microbody targeting signal" (ECO:0000255) conflicts with in vivo evidence that the C-terminal SRL is dispensable and that an N-terminal PTS2 directs import (PMID:8511963, PMID:7805876). Should the record be revised to annotate the N-terminal PTS2?
Q: Which H. polymorpha copper amine oxidase (AMO/HPAO-1 or HPAO-2) carries out the peroxisomal amine oxidation seen during choline and ethanolamine degradation?
Experiment: Delete AMO and HPAO-2 singly and together in H. polymorpha, then measure growth on methylamine, ethylamine, benzylamine and choline as sole nitrogen source, with complementation by wild-type and D319N AMO.
Type: genetic
Experiment: Map the PTS2 within the first 16 residues of AMO by point mutation of the consensus residues, and test Pex7 dependence of import in pex7 cells.
Type: cell biology
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