AmpDh2 (PA5485) is one of three AmpD paralogues of Pseudomonas aeruginosa, a zinc-dependent N-acetylmuramoyl-L-alanine amidase of the Amidase_2 family with an additional peptidoglycan-binding domain (PG_binding_1). It is a lipoprotein with a signal peptide and lipobox, a dimer that inserts into the inner leaflet of the outer membrane and removes stem peptides from the peptidoglycan sacculus, cutting both cross-linked and non-cross-linked stems; it works at the periphery of the sacculus, releasing mostly soluble products. Purified AmpDh2 prefers normal muramyl peptides (cell-wall surrogates) and has only marginal activity on the 1,6-anhydromuramyl recycling metabolites that are the substrates of cytoplasmic AmpD, so its role is cell-wall remodelling or maturation rather than recycling. That marginal activity on periplasmic anhydromuropeptides is thought to explain why ampDh2 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). ampDh2 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 ampDh2 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 Overnight incubation of the polymeric sacculus with AmpDh2 produced products with oligomeric cell-wall sugars without the peptide stem. |
| 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 Overnight incubation of the polymeric sacculus with AmpDh2 produced products with oligomeric cell-wall sugars without the peptide stem. |
| GO:0009253 peptidoglycan catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping. ampDh2 removes stem peptides from polymeric peptidoglycan, breaking down the cell-wall polymer. Reason: Direct participation; the enzyme degrades peptidoglycan. Supporting Evidence: PMID:23510438 Overnight incubation of the polymeric sacculus with AmpDh2 produced products with oligomeric cell-wall sugars without the peptide stem. |
| GO:0009254 peptidoglycan turnover | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN001252273, seeded by E. coli AmpD. For ampDh2 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 | ACCEPT | Summary: IBA from the PAINT IBD on PTN002019137 (seeds ampD, ampDh2, ampDh3). For AmpDh2 the claim holds: it is an outer-membrane lipoprotein that acts on the sacculus. At node level the IBD is unsound, because the clade includes the cytoplasmic AmpD lineage and the ampD seed is wrong; the GO_CENTRAL defect is recorded on the ampD review. Reason: True for this gene, which acts on the peptidoglycan layer from the inner leaflet of the outer membrane; the node-level problem affects other leaves, not AmpDh2. Supporting Evidence: PMID:23931161 AmpDh2 is a dimeric protein that inserts itself into the inner leaflet of the outer membrane, where it interacts and hydrolyzes the amide bond between the stem peptide and the NAM moiety of the sacculus. |
| GO:0009276 Gram-negative-bacterium-type cell wall | IDA PMID:23510438 Reactions of the three AmpD enzymes of Pseudomonas aeruginos... | ACCEPT | Summary: PseudoCAP IDA and a seed of the PTN002019137 IBD. The paper did not localize the protein; it cites earlier work for the signal peptide and outer-membrane anchor and shows activity on isolated sacculus. The claim is correct (outer-membrane lipoprotein acting on peptidoglycan), but IDA overstates the evidence. Reason: The location is right, supported by the lipoprotein signal and by the laboratory's later structural work. The evidence code is a source issue, recorded as a defect. Supporting Evidence: PMID:23510438 AmpDh2 has a signal peptide that targets it to the periplasm and an anchor that is believed to insert it into the outer membrane. PMID:23931161 AmpDh2 is a dimeric protein that inserts itself into the inner leaflet of the outer membrane, where it interacts and hydrolyzes the amide bond between the stem peptide and the NAM moiety of the sacculus. |
| 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 ampDh2 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 ampDh2 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. |
| GO:0009279 cell outer membrane | IC PMID:23931161 Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudo... | NEW | Summary: Proposed outer-membrane location. Two independent lines agree: the sequence has a SignalP signal peptide and a PROSITE prokaryotic lipoprotein lipid-attachment site, and the laboratory that characterised the enzyme reports that the dimer inserts into the inner leaflet of the outer membrane. Reason: Adds the membrane on which this lipoprotein is anchored, next to the cell-wall rows; consistent with the E. coli comparator AmiD, an outer-membrane lipoprotein. Supporting Evidence: PMID:23931161 AmpDh2 is a dimeric protein that inserts itself into the inner leaflet of the outer membrane, where it interacts and hydrolyzes the amide bond between the stem peptide and the NAM moiety of the sacculus. PMID:23510438 AmpDh2 has a signal peptide that targets it to the periplasm and an anchor that is believed to insert it into the outer membrane. |
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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)?
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