AmpDh3 (PA0807) is one of three AmpD paralogues of Pseudomonas aeruginosa, a zinc-dependent N-acetylmuramoyl-L-alanine amidase of the Amidase_2 family with a peptidoglycan-binding-like domain. Purified AmpDh3 is a tetramer that removes the stem peptides from peptidoglycan, acting mainly on the insoluble polymeric sacculus in a processive, multivalent manner, and it prefers normal muramyl peptides (cell-wall surrogates) to the 1,6-anhydromuramyl recycling metabolites of cytoplasmic AmpD, so its role is cell-wall remodelling rather than recycling. Its cellular location is unsettled: it has been described as periplasmic, but it has no predicted signal peptide, other workers call it cytosolic, and a reported role as a type VI secretion effector was not reproduced. Its marginal activity on anhydromuropeptides is thought to explain why ampDh3 loss adds to the AmpC beta-lactamase derepression of ampD mutants.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the PAINT IBD on PTN001252273 (5 seeds, including the characterised E. coli AmpD and AmiD). ampDh3 is inside this Amidase_2 clade and is itself a seed, so its appearance in WITH/FROM is expected. Node assessment in the ampD notes. Reason: Core activity, shown directly with purified enzyme. Supporting Evidence: PMID:23510438 Hence, it is clear that AmpDh2 and AmpDh3 have evolved as enzymes for processing of the peptidoglycan components of the cell wall in the periplasmic space. |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IDA PMID:23510438 Reactions of the three AmpD enzymes of Pseudomonas aeruginos... | ACCEPT | Summary: PseudoCAP IDA and a seed of the PTN001252273 IBD. Purified ampDh3 hydrolyses the lactyl-amide bond of muramyl peptides and strips stem peptides from isolated sacculus, with kinetics measured for six substrates. Reason: The seed is sound; direct enzymology for the core activity. Supporting Evidence: PMID:23510438 Hence, it is clear that AmpDh2 and AmpDh3 have evolved as enzymes for processing of the peptidoglycan components of the cell wall in the periplasmic space. PMID:23510438 The reaction with AmpDh3 also produced products (20), but its quantity was three times less than that with AmpDh2. |
| GO:0008745 N-acetylmuramoyl-L-alanine amidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Amidase_2 domain and EC mapping; same claim as the IDA row. Reason: Consistent with the direct biochemical evidence. Supporting Evidence: PMID:23510438 The reaction with AmpDh3 also produced products (20), but its quantity was three times less than that with AmpDh2. |
| GO:0009253 peptidoglycan catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping. ampDh3 removes stem peptides from polymeric peptidoglycan, breaking down the cell-wall polymer. Reason: Direct participation; the enzyme degrades peptidoglycan. Supporting Evidence: PMID:23510438 The reaction with AmpDh3 also produced products (20), but its quantity was three times less than that with AmpDh2. |
| GO:0009254 peptidoglycan turnover | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN001252273, seeded by E. coli AmpD. For ampDh3 the process is cell-wall remodelling (turnover of the sacculus itself) rather than recycling of imported fragments, but both fall under peptidoglycan turnover. Reason: Core process; the paper identifies the paralogue as a cell-wall turnover enzyme. Supporting Evidence: PMID:23510438 AmpDh2 and AmpDh3 as enzymes involved in turnover of the bacterial cell wall itself PMID:23931161 The present study documents that AmpDh2 and AmpDh3 complement each other in remodeling of the cell wall. |
| GO:0009276 Gram-negative-bacterium-type cell wall | IBA GO_REF:0000033 | UNDECIDED | Summary: IBA from the PAINT IBD on PTN002019137 (seeds ampD, ampDh2, ampDh3). The node is unsound (it includes the cytoplasmic AmpD lineage and the ampD seed is wrong; the GO_CENTRAL defect is recorded on the ampD review). For AmpDh3 itself the location is unsettled. It acts on the sacculus in vitro and its own laboratory calls it periplasmic, but it has no predicted signal peptide, the seed paper says its location is unknown, and a later study found no export and describes it as cytosolic. Reason: Conflicting statements on the location and no fractionation data; the IBA adds nothing beyond this gene's own uncertain seed and AmpDh2. Propagation Review Root cause: UNRESOLVED Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN002019137 Β· PTN002019137 SUPPORTS SOURCE BUT NOT TARGET Node mixes cytoplasmic AmpD orthologues with periplasmic or outer-membrane AmiD and AmpDh2; node-level GO_CENTRAL defect recorded on the ampD review. UniProtKB:Q9I5D1 Β· ampDh3 (self; PseudoCAP IDA PMID:23510438) SOURCE WEAK OR INFERRED Location inferred from substrate preference, not assayed. UniProtKB:G3XCW9 Β· ampD SOURCE BAD Cytoplasmic enzyme; the cell-wall seed row is wrong. UniProtKB:Q9HT86 Β· ampDh2 SUPPORTS SOURCE BUT NOT TARGET Outer-membrane lipoprotein; its location does not transfer to a protein without a signal peptide. Supporting Evidence: PMID:23510438 the cellular location of AmpDh3 is currently unknown PMID:36577706 we found no evidence that AmpDh3 is exported from cells |
| GO:0009276 Gram-negative-bacterium-type cell wall | IDA PMID:23510438 Reactions of the three AmpD enzymes of Pseudomonas aeruginos... | UNDECIDED | Summary: PseudoCAP IDA and a seed of the PTN002019137 IBD. The paper (full text read) did not localize AmpDh3; it states that its location is unknown and proposes a periplasmic role from its preference for cell-wall substrates. Location is inferred from in vitro activity, not observed. Reason: The paper does not support the location as IDA, and the literature disagrees on whether AmpDh3 is periplasmic (no signal peptide; not exported in a later study). The claim may be true, but there is no direct localization to accept it on. Supporting Evidence: PMID:23510438 the cellular location of AmpDh3 is currently unknown PMID:23931161 AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa PMID:36577706 we found no evidence that AmpDh3 is exported from cells |
| GO:0033252 regulation of beta-lactamase activity | IDA PMID:23510438 Reactions of the three AmpD enzymes of Pseudomonas aeruginos... | REMOVE | Summary: The paper did no beta-lactamase assay. It proposes that the marginal activity of ampDh3 on periplasmic anhydromuropeptides explains the stepwise AmpC derepression seen in earlier genetic studies. That is an indirect effect on ampC induction via the AmpR inducer pool, and GO:0033252 is about modulating beta-lactamase catalytic activity, which ampDh3 does not do. Reason: Wrong term and no direct evidence; the effect is on AmpC expression, indirectly, through inducer turnover. Supporting Evidence: PMID:23510438 Nonetheless, the marginal activity in turnover of metabolite 1 with AmpDh2 and AmpDh3 explains the stepwise effect observed in augmenting derepression of AmpC Ξ²-lactamase by eliminations of these enzymes one-by-one. |
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Download this section (compressed HTML)Q: PTN002019137 carries GO:0009276 for a clade that includes the cytoplasmic AmpD lineage. Should the IBD be moved to the AmiD/AmpDh2/AmpDh3 subclade (see the ampD review)?
Q: How does AmpDh3, which has no predicted signal peptide, reach the sacculus? Is it periplasmic, cytoplasmic, or exported by a non-classical route?
Experiment: Subcellular fractionation of P. aeruginosa expressing chromosomally tagged AmpDh3, with cytoplasmic, periplasmic and membrane markers, and a test of whether periplasmic targeting is needed for its contribution to AmpC derepression.
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