lap (PA2939; PaAP, also called PaaP or LAP) encodes the major secreted aminopeptidase of Pseudomonas aeruginosa, a peptidase M28 (M28A, SGAP-like) family enzyme with an N-terminal protease-associated (PA) domain and a binuclear zinc catalytic domain. It is made as a 536-residue pre-proenzyme: the signal peptide is removed on export across the inner membrane, the proenzyme is secreted through the Xcp type II secretion system, and it is activated outside the cell when the endopeptidase LysC (and, less directly, elastase and alkaline protease) removes an autoinhibitory C-terminal propeptide that otherwise blocks the active-site cleft. The mature enzyme removes single N-terminal residues from peptides and unstructured protein regions that carry a free amino terminus, with a preference for Leu and Lys; it is thermostable, has a pH optimum near 8.5 and is inhibited by metal chelators. PaAP is one of the most abundant proteins of the biofilm matrix and of outer membrane vesicles. It completes the extracellular digestion of proteins begun by the secreted endopeptidases, releasing amino acids that support growth on protein (an RpoS- and LasR-regulated public good), and its activity modulates biofilm development and dispersal.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004177 aminopeptidase activity | EXP PMID:11533066 A secreted aminopeptidase of Pseudomonas aeruginosa. Identif... | MODIFY | Summary: Aminopeptidase activity of purified PaAP (AP56 and AP28) on Leu-pNA and on peptides with a free N-terminus was shown directly in Cahan et al. 2001. Reason: The activity is well established, but the term is less specific than the evidence. The same paper shows inhibition by Zn chelators but not serine or thiol protease inhibitors, and the crystal structures (PMID:37386135) show a binuclear zinc active site, so the appropriate term is the is_a descendant metalloaminopeptidase activity (GO:0070006), which also subsumes the InterPro-derived metalloexopeptidase activity row. Proposed replacements: metalloaminopeptidase activity Supporting Evidence: PMID:11533066 This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. PMID:37386135 The active site contains two zinc ions; Zn1 is coordinated by D306, H296 and D369, while Zn2 is coordinated by E341, H467 and D306. file:PSEAE/lap/lap-uniprot.txt A secreted aminopeptidase. Acts on free N-terminal amino groups with a very strong preference for Leu- followed by Met- and Ala- |
| GO:0004177 aminopeptidase activity | IEA GO_REF:0000120 | MODIFY | Summary: Electronic aminopeptidase annotation from the M28 SGAP-like InterPro signature and the EC number; correct and consistent with the experimental row. Reason: Same claim as the EXP row; refined in the same way to metalloaminopeptidase activity, which the M28 binuclear zinc architecture and the chelator sensitivity support. Proposed replacements: metalloaminopeptidase activity Supporting Evidence: file:PSEAE/lap/lap-uniprot.txt Belongs to the peptidase M28 family. M28A subfamily. PMID:37386135 PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. |
| GO:0005576 extracellular region | EXP PMID:11533066 A secreted aminopeptidase of Pseudomonas aeruginosa. Identif... | ACCEPT | Summary: Activity and the AP56 protein were recovered from culture filtrates of several strains; PaAP is a secreted enzyme that acts outside the cell. Reason: Direct evidence from culture filtrates; the extracellular location is where the core activity is exerted (the enzyme is activated and acts extracellularly). Supporting Evidence: PMID:11533066 we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains PMID:30047246 PaAP functioned extracellularly and aminopeptidase catalytic activity was essential for its effect on biofilm development. |
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the UniProt "Secreted" subcellular location, which is itself experimentally backed (ECO:0000269, PMID:11533066). Reason: Consistent with the experimental row; correct location. Supporting Evidence: file:PSEAE/lap/lap-uniprot.txt SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:11533066}. |
| GO:0006508 proteolysis | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PAINT node PTN000254534 (PTHR12147, peptidase M28), whose IBD is seeded only by this gene's own PseudoCAP IDA row. The target appearing in its own WITH/FROM is expected, not circular. Reason: The seed is sound (direct peptide hydrolysis by the purified enzyme), PaAP retains the conserved M28 zinc ligands and is catalytically active, so it is squarely inside the clade that inherits peptidase function. Proteolysis is the appropriate general process term for a peptidase; the more specific information belongs in the MF (metalloaminopeptidase activity). Supporting Evidence: PMID:39126017 Leu-Ala and Ala4, having free N-termini, are both cleaved readily [17]. PMID:37386135 PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. |
| GO:0006508 proteolysis | IDA PMID:11533066 A secreted aminopeptidase of Pseudomonas aeruginosa. Identif... | ACCEPT | Summary: PseudoCAP IDA; sole seed of the PAINT IBD at PTN000254534 (272 leaf IBAs in 109 taxa). Cahan et al. purified PaAP from culture filtrates and assayed its hydrolysis of amino-acid p-nitroanilides and of short peptides. Reason: Sound seed. The paper directly measures peptide-bond hydrolysis by the gene product (Leu-pNA; Leu-Ala and Ala4 with free N-termini, per the author's 2024 review and UniProt's ECO:0000269 curation from the full text), so the enzyme itself performs the process. The cached text is abstract-only, but the abstract alone documents the enzyme assay and the corroborating sources agree. Supporting Evidence: PMID:11533066 Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. PMID:39126017 Leu-Ala and Ala4, having free N-termini, are both cleaved readily [17]. |
| GO:0006508 proteolysis | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO proteolysis from the M28 family signatures; correct. Reason: Consistent with the experimental and IBA rows for the same term. Supporting Evidence: file:PSEAE/lap/lap-uniprot.txt Belongs to the peptidase M28 family. M28A subfamily. |
| GO:0008235 metalloexopeptidase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO metalloexopeptidase activity from the M28 family signature; correct, as PaAP is a zinc-dependent exopeptidase. Reason: Correct but general. The exopeptidase is specifically an aminopeptidase, so the is_a descendant metalloaminopeptidase activity (GO:0070006) captures both this row and the aminopeptidase rows. Proposed replacements: metalloaminopeptidase activity Supporting Evidence: PMID:37386135 The active site contains two zinc ions; Zn1 is coordinated by D306, H296 and D369, while Zn2 is coordinated by E341, H467 and D306. |
| GO:0015628 protein secretion by the type II secretion system | IDA PMID:9642203 Secretion of elastinolytic enzymes and their propeptides by ... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IDA from Braun et al. 1998, who detected the then-uncharacterised SGAP homologue in the culture medium of protease-deficient strains. PaAP is indeed exported by the Xcp T2SS, but as cargo. Reason: The biology (PaAP crosses the outer membrane via Xcp) is accepted, but a secreted substrate does none of the work of the secretion process; GO annotates the T2SS machinery (Xcp/Gsp proteins) to this term, not their cargo. The fact belongs on the transporter side (has_input / cargo) and in the cellular-component and description fields here. Full text of PMID:9642203 was not available (PMC restricts access), so the precise experiment behind the row could not be checked; I therefore do not REMOVE and record the issue as a probable source defect. Supporting Evidence: PMID:39126017 the resulting pro-PaAP is secreted across the outer membrane via the Xcp export machinery (step 2) [23] PMID:9642203 Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions. |
| GO:0043952 protein transport by the Sec complex | IDA PMID:9642203 Secretion of elastinolytic enzymes and their propeptides by ... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IDA from Braun et al. 1998; the only experimental fact visible for PaAP is removal of its signal peptide (UniProt: protein sequence of residues 25-34 from this paper). Reason: Two problems. First, PaAP is a cargo and does not perform Sec-dependent translocation (SecYEG/SecA do). Second, signal-peptide removal shows export across the inner membrane but does not by itself identify the Sec translocon (versus Tat). Full text was not available, so whether the paper has Sec-specific evidence is unchecked; I do not REMOVE, and flag a probable PseudoCAP defect on the cargo point. Supporting Evidence: file:PSEAE/lap/lap-uniprot.txt PROTEIN SEQUENCE OF 25-34. PMID:9642203 The signal sequence is cleaved off during transport across the inner membrane |
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Download this section (compressed HTML)Q: PTN000254534 carries the only IBD in PTHR12147 (proteolysis), seeded solely by PaAP. If the node spans only SGAP-like M28A aminopeptidases, could PAINT add a metalloaminopeptidase activity (GO:0070006) IBD, seeded also by characterised members such as Streptomyces griseus aminopeptidase (P80561) and B. subtilis YwaD (P25152)?
Q: Should the Xcp T2SS cargo relationship for PaAP be captured as has_input on the Xcp machinery (e.g. in a GO-CAM) rather than as a PseudoCAP process annotation on the cargo?
Q: Does the PA domain of mature PaAP recruit protein substrates or mediate its association with outer membrane vesicles?
Experiment: Systematic N-terminomics of protein and peptide substrates (and OMV-bound versus free enzyme) with wild type and PA-domain-deleted PaAP, to define the natural substrate range and the role of the PA domain.
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