AmpD (PA4522) is the cytoplasmic zinc-dependent 1,6-anhydro-N-acetylmuramyl-L-alanine amidase of Pseudomonas aeruginosa, a member of the N-acetylmuramoyl-L-alanine amidase 2 (Amidase_2/PGRP) family. In cell-wall recycling it removes the stem peptide from 1,6-anhydromuramyl peptides that are generated in the periplasm by lytic transglycosylases, imported by AmpG and trimmed by NagZ. Purified AmpD hydrolyses the lactyl-amide bond of anhydromuramyl tri-, tetra- and pentapeptides but not of the corresponding normal muramyl peptides, so it is specific for recycling metabolites. By destroying the anhydromuropeptide that activates the transcriptional regulator AmpR, AmpD keeps expression of the chromosomal AmpC beta-lactamase low; ampD loss causes partial AmpC derepression, which becomes full when the periplasmic paralogues AmpDh2 and AmpDh3 are also lost. ampD forms an operon with ampE.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping. The UniProt location line is ARBA-derived, but the claim is independently sound. AmpD has no signal peptide, its substrates (anhydromuramyl peptides produced by NagZ after AmpG import) exist only in the cytoplasm, and the biochemical study identifies it as the cytoplasmic paralogue. The E. coli orthologue P13016 is cytoplasmic. Reason: Location where the core activity acts, supported by the substrate logic of the recycling pathway and by the orthologue. Supporting Evidence: PMID:23510438 This analysis identifies AmpD of P. aeruginosa as the cytoplasmic protease for commitment to the cell-wall recycling events and for the reversal of the activation of the antibiotic-resistance pathway. file:PSEAE/ampD/ampD-bioinformatics/P13016.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305|PubMed:19309146}. |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the PAINT IBD on PTN001252273 (5 seeds: E. coli AmpD and AmiD and the three P. aeruginosa AmpD paralogues). The clade is the Amidase_2 family of PTHR30417, whose members cleave the MurNAc-L-Ala bond. ampD is inside the clade and its own seeding appears in WITH/FROM as expected. Reason: Core activity, confirmed for this gene by kinetics with purified enzyme. Supporting Evidence: PMID:23510438 AmpD is the true protease for the recycling process in P. aeruginosa, as its function is exclusive for turnover of 1,6-anhydromuramyl variants 4–6 |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined InterPro (Amidase_2 domain) and EC mapping; same claim as the IBA and IMP rows. Reason: Consistent with the direct biochemical evidence. Supporting Evidence: PMID:23510438 the nature of the enzymatic reaction of AmpD with this compound is hydrolysis of the amide bond to the lactyl moiety |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IMP PMID:9835532 An ampD gene in Pseudomonas aeruginosa encodes a negative re... | ACCEPT | Summary: PseudoCAP IMP and a seed of the PTN001252273 IBD. The abstract reports that P. aeruginosa ampD restores wild-type beta-lactamase expression to E. coli ampD mutants, a functional complementation of the amidase. Later biochemistry (PMID:23510438) confirmed the activity with purified enzyme. Reason: The seed is sound. Heterologous complementation of the amidase defect is genetic support for the activity, and the term is confirmed directly by kinetics. Supporting Evidence: PMID:9835532 The ampD product transcomplemented Escherichia coli ampD mutants to wild-type beta-lactamase expression. PMID:23510438 AmpD is the true protease for the recycling process in P. aeruginosa, as its function is exclusive for turnover of 1,6-anhydromuramyl variants 4–6 |
| GO:0009253 peptidoglycan catabolic process | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro mapping. AmpD hydrolyses peptidoglycan-derived anhydromuropeptides, a step in the breakdown of released cell-wall fragments. Reason: True, but AmpD acts on soluble recycling intermediates rather than on the sacculus; peptidoglycan turnover (recycling) better describes its role. Supporting Evidence: PMID:23510438 AmpD is the true protease for the recycling process in P. aeruginosa, as its function is exclusive for turnover of 1,6-anhydromuramyl variants 4–6 |
| GO:0009254 peptidoglycan turnover | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN001252273, seeded only by E. coli AmpD. Cell-wall recycling, in which AmpD performs the peptide-release step, is part of peptidoglycan turnover. Reason: Core process; AmpD catalyses a step of peptidoglycan recycling. Supporting Evidence: PMID:23510438 This analysis identifies AmpD of P. aeruginosa as the cytoplasmic protease for commitment to the cell-wall recycling events and for the reversal of the activation of the antibiotic-resistance pathway. |
| GO:0009276 Gram-negative-bacterium-type cell wall | IBA GO_REF:0000033 | REMOVE | Summary: IBA from the PAINT IBD on PTN002019137, whose only seeds are the three P. aeruginosa AmpD paralogues. The clade also contains E. coli AmpD (P13016) and the AmpD orthologues of H. influenzae, Salmonella, Yersinia, Vibrio, Coxiella and Shewanella, which are cytoplasmic recycling enzymes. AmpD itself is cytoplasmic and acts on imported anhydromuropeptides, not on the peptidoglycan layer. Reason: Wrong location for this gene. The IBA rests partly on the wrong PseudoCAP seed below, and the remaining seeds (periplasmic AmpDh2 and AmpDh3) do not justify placing a cell-wall location at a node that includes the cytoplasmic AmpD lineage. Propagation Review Root cause: SOURCE BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH SOURCE EVIDENCE WEAK Sources checked: PANTHER:PTN002019137 · PTN002019137 SUPPORTS SOURCE BUT NOT TARGET 18 leaves in 11 taxa, mixing cytoplasmic AmpD orthologues (E. coli P13016, H. influenzae P44624, Salmonella P0CL03, Yersinia Q0WBM3, Vibrio Q9KPE7 and others) with periplasmic or outer-membrane paralogues (AmiD P75820, AmpDh2, AmpDh3). A cell-wall location does not suit the whole clade. UniProtKB:G3XCW9 · ampD (self; PseudoCAP IMP PMID:9835532) SOURCE BAD The self-seed is the wrong PseudoCAP cell-wall row; a complementation of beta-lactamase expression does not localize the protein. UniProtKB:Q9HT86 · ampDh2 SUPPORTS SOURCE BUT NOT TARGET Periplasmic outer-membrane lipoprotein acting on the sacculus; true for it. UniProtKB:Q9I5D1 · ampDh3 SUPPORTS SOURCE BUT NOT TARGET Acts on the sacculus; true for it, but not for the AmpD lineage. Supporting Evidence: PMID:23510438 This analysis identifies AmpD of P. aeruginosa as the cytoplasmic protease for commitment to the cell-wall recycling events and for the reversal of the activation of the antibiotic-resistance pathway. file:PSEAE/ampD/ampD-bioinformatics/P13016.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000305|PubMed:19309146}. |
| GO:0009276 Gram-negative-bacterium-type cell wall | IMP PMID:9835532 An ampD gene in Pseudomonas aeruginosa encodes a negative re... | REMOVE | Summary: PseudoCAP IMP and a seed of the PTN002019137 IBD. A complementation of beta-lactamase expression in E. coli ampD mutants does not localize the protein, and AmpD is the cytoplasmic member of the three paralogues. Reason: Wrong location; the reported mutant phenotype does not address a cellular component, and the biochemistry places AmpD in the cytoplasm. Supporting Evidence: PMID:23510438 This analysis identifies AmpD of P. aeruginosa as the cytoplasmic protease for commitment to the cell-wall recycling events and for the reversal of the activation of the antibiotic-resistance pathway. |
| GO:0033252 regulation of beta-lactamase activity | IMP PMID:9835532 An ampD gene in Pseudomonas aeruginosa encodes a negative re... | REMOVE | Summary: AmpD lowers AmpC beta-lactamase production by degrading the anhydromuropeptide that activates AmpR, which controls ampC transcription. The paper measured beta-lactamase expression. GO:0033252 is defined as modulation of the beta-lactamase catalytic activity (is_a regulation of hydrolase activity), which AmpD does not do. Reason: Wrong term; AmpD affects ampC expression indirectly, through the AmpR inducer, and not the enzymatic activity of AmpC. No replacement process term is proposed, because AmpD's part in AmpR-dependent induction is metabolic (inducer turnover). Supporting Evidence: PMID:9835532 The ampD product transcomplemented Escherichia coli ampD mutants to wild-type beta-lactamase expression. |
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Download this section (compressed HTML)Q: GO:0009392 N-acetyl-anhydromuramoyl-L-alanine amidase activity names the anhydro-specific reaction of AmpD but is not placed under GO:0008745. Should it be made an is_a child of GO:0008745, and should its definition cover the anhydromuramyl peptides (without GlcNAc) that AmpD hydrolyses after NagZ action?
Q: Which subclade of PTN002019137 should carry the cell-wall location, given that the node mixes cytoplasmic AmpD orthologues with periplasmic AmiD, AmpDh2 and AmpDh3?
Experiment: Fractionate P. aeruginosa expressing chromosomally tagged AmpD, AmpDh2 and AmpDh3, with cytoplasmic, periplasmic and outer-membrane markers, to confirm the location of each paralogue directly.
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