lap (PA2939, Q9HZQ8) — PaAP secreted aminopeptidase — review notes

No deep-research provider was available; the notes below are from my own
reading of the cached publications (publications/PMID_*.md) and the UniProt
record (lap-uniprot.txt).

Identity

UniProt Q9HZQ8 (LAP_PSEAE), 536 aa precursor, signal peptide 1-24, peptidase
M28 family, M28A subfamily; PA domain + M28 Zn-peptidase domain. PANTHER
PTHR12147:SF26. MEROPS M28.003/M28.008. Crystal structures 8AC7, 8AC9, 8ACG,
8ACK, 8ACR.
Names: PaAP, LAP, PaaP (Zhao et al. 2018 named the gene paaP)
PMID:30047246.

Activity

Discovery: aminopeptidase activity on Leu-pNA in culture filtrates; AP56
converted to active AP28 by heating with elastase; Zn-chelator sensitive;
N-terminal sequences matched PA2939 [PMID:11533066 "Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated
aminopeptidase activity in the culture filtrates of several Pseudomonas
aeruginosa strains"; "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"]. Abstract only in cache; UniProt
curated CC FUNCTION/CATALYTIC ACTIVITY from the full text (ECO:0000269) adds
that peptides with a free N-terminus are hydrolysed (Leu > Met > Ala).
Per the 2024 review, Cahan et al. also showed hydrolysis of Leu-Ala and Ala4
(free N-termini) and no cleavage of N-blocked peptides
PMID:39126017.
This is direct evidence of peptide-bond hydrolysis in peptides, i.e. the
basis of the PseudoCAP IDA "proteolysis" row.
Promiscuous aminopeptidase on unstructured regions of peptides and proteins;
autoinhibited by its C-terminal propeptide PMID:37386135.
Binuclear zinc active site confirmed by crystal structure
PMID:37386135
(note: the 2024 review numbers the first Asp as 308; I do not rely on the
residue number). So the specific MF is metalloaminopeptidase activity
(GO:0070006, is_a aminopeptidase activity and is_a metalloexopeptidase activity).
Lys-pNA and Leu-pNA are best substrates; AP56 and AP28 have the same kinetics
on small substrates PMID:34133429.
EC: UniProt gives EC 3.4.11.1 (leucyl aminopeptidase). EC 3.4.11.1 is formally
the M17 cytosol aminopeptidase; PaAP is an M28 SGAP-like enzyme. I therefore do
not propose the EC-linked GO term and stay at metalloaminopeptidase activity.

Activation, secretion, location

Secreted via the Xcp type II secretion system, activated extracellularly by
LysC cleavage of the C-terminal propeptide (Lys512-Ala513) PMID:34133429; review PMID:39126017.
Braun et al. 1998 (PMID:9642203) found the uncharacterised SGAP homologue
accumulating in culture medium of protease-deficient strains; N-terminal
sequence (residues 25-34) shows the signal peptide removed. Full text not
available (PMC restricts). The 2024 review cites it as the source for Xcp-dependent
outer membrane passage PMID:39126017.
Extracellular; abundant in biofilm matrix and in outer membrane vesicles,
especially from CF isolates PMID:16807039.

Biology

Nutrient acquisition: completes extracellular protein digestion begun by
endopeptidases; RpoS-controlled public good PMID:32184248.
Biofilm modulation: deletion increases early attachment, late cell death and
biofilm disruption; catalytic activity required PMID:30047246; OMV-associated anti-biofilm/detachment
activity PMID:31744920. These are pleiotropic
phenotypes downstream of nutrient supply; I do not propose biofilm process terms.
Regulation: LasR/LasI quorum sensing and RpoS (principal activator) PMID:39126017.

Annotation decisions (summary)

Aminopeptidase activity (EXP, IEA) and metalloexopeptidase activity (IEA):
correct but general; MODIFY to metalloaminopeptidase activity GO:0070006
(Zn-chelator inhibition + binuclear Zn site in the crystal structure).
Proteolysis (IDA PseudoCAP, IBA, IEA): ACCEPT. Process-level term for a
peptidase; consistent across rows.
Extracellular region (EXP, IEA): ACCEPT.
Protein secretion by the T2SS and protein transport by the Sec complex (IDA
PseudoCAP, PMID:9642203): PaAP is the cargo, not a component of either
machine. Under GO practice, cargo is not annotated as participating in its own
transport process (the transporter does the work). MARK_AS_OVER_ANNOTATED, with
a PROBABLE PseudoCAP source defect (the cargo role is the positive basis).
The only evidence we could see for the Sec row is signal-peptide removal
(N-terminal sequencing), which does not by itself identify the Sec
translocon. Full text of PMID:9642203 was not available (PubMed MCP and
Europe PMC returned no body text, 2026-10-29), so whether it holds
Sec-specific evidence is unchecked.

PseudoCAP IBA-seed assessment

PTN node	node term	n_seeds	leaf IBAs	seed annotation (term, evidence, PMID)	verdict	rationale
PTN000254534	GO:0006508 proteolysis	1 (sole seed)	272 (109 taxa)	GO:0006508 proteolysis, IDA, PMID:11533066 (PseudoCAP)	SOUND	Cahan et al. purified the enzyme and showed Zn-dependent hydrolysis of amino-acid p-nitroanilides and of peptides with free N-termini (Leu-Ala, Ala4), i.e. direct peptide-bond hydrolysis; the gene product does the work. Abstract only in cache, but the UniProt ECO:0000269 curation and the 2024 review corroborate the full-text content. The node term is deliberately general: PTHR12147 M28 members (SGAP-like aminopeptidases, fungal "probable zinc metalloproteases", Mtb LpqL, E. coli YfbL) are peptidases with the conserved Zn ligands, so "proteolysis" is safe at the node. It is the only IBD in the whole family (PTHR12147-paint.tsv), so the clade carries no MF; a metalloaminopeptidase activity IBD would be more informative, but needs seeds beyond PaAP (e.g. SGAP P80561, B. subtilis YwaD P25152) and a check of the node's membership.

Non-seeding PseudoCAP rows on this gene (T2SS secretion, Sec transport;
PMID:9642203) are cargo annotations; see above.

Open questions

Which subtree does PTN000254534 span? If it covers only bacterial SGAP-like
M28A enzymes, PAINT could add an MF IBD (metalloaminopeptidase activity) using
SGAP/YwaD/PaAP as seeds.
Is the PA domain involved in protein-substrate recruitment or OMV association?