PA0845 (cerN) is a secreted neutral/alkaline ceramidase (N-acylsphingosine amidohydrolase, EC 3.5.1.23) of Pseudomonas aeruginosa. It hydrolyses the N-acyl linkage of ceramides to release sphingosine and a free fatty acid at alkaline pH, and catalyses the reverse condensation in vitro; it is a zinc-dependent metalloenzyme and acts on a broad range of ceramides. In the lung, cerN cleaves host ceramide to sphingosine, which the AraC-family regulator SphR senses to induce the sphingosine catabolic (sph) regulon; cerN is itself a member of that SphR regulon. It is the founding member of the neutral ceramidase family, with homologues in mammals, fungi, insects and other bacteria.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005576 extracellular region | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location (extracellular region). The enzyme was purified from culture supernatant and has a signal peptide. Reason: Secreted enzyme. Supporting Evidence: PMID:9603946 it hydrolyzed ceramides containing C12:0-C18:0 fatty acids |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN004377481, the neutral ceramidase family (156 leaves, 78 taxa). Seven seeds: PA0845 (self), human ASAH2 (Q9NR71), mouse, rat, zebrafish, fly and M. tuberculosis neutral ceramidase (O06769, IDA PMID:10593963/20139604). Stands without this seed. Reason: Core activity; directly assayed for the P. aeruginosa enzyme. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. PMID:10593963 Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IDA PMID:9603946 Purification and characterization of a novel ceramidase from... | ACCEPT | Summary: UniProt IDA: purified enzyme hydrolyses ceramides. Reason: Direct biochemical evidence. Supporting Evidence: PMID:9603946 This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC/Rhea mapping to the same activity. Reason: Consistent with the experimental rows. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. |
| GO:0017040 N-acylsphingosine amidohydrolase activity | IMP PMID:10593963 Molecular cloning, sequencing, and expression of the gene en... | ACCEPT | Summary: PseudoCAP seed row for PTN004377481. Seed verdict SOUND: the gene was cloned and the recombinant enzyme confirmed to be an active ceramidase. Reason: Recombinant expression confirms the activity. Supporting Evidence: PMID:10593963 Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. |
| GO:0042759 long-chain fatty acid biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from PTN004377481 for long-chain fatty acid biosynthetic process, seeded only by PA0845 and M. tuberculosis O06769. A ceramidase releases a free fatty acid by hydrolysis; it does not synthesise one. Reason: The free fatty acid is a hydrolysis product of ceramide cleavage, not a biosynthetic product. Over-annotation; see the GO_CENTRAL defect on the IDA row. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN004377481 · PTN004377481 SOURCE BAD Node term treats a hydrolytic release of fatty acid as biosynthesis. |
| GO:0042759 long-chain fatty acid biosynthetic process | IDA PMID:9603946 Purification and characterization of a novel ceramidase from... | MARK AS OVER ANNOTATED | Summary: UniProt IDA from PMID:9603946, the same misreading: the purified enzyme releases a free fatty acid by hydrolysis. Reason: Releasing a fatty acid by ceramide hydrolysis is not fatty-acid biosynthesis. Node-level GO_CENTRAL defect recorded here, on the gene with the most direct evidence for this node. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. |
| GO:0046512 sphingosine biosynthetic process | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from PTN004377481 for sphingosine biosynthetic process (seeds include mammalian ceramidases and PA0845). The enzyme produces sphingosine by cleaving ceramide. Reason: Sphingosine is a genuine product of the reaction and this is a conserved family annotation, but in P. aeruginosa the enzyme's role is ceramide catabolism and host-sphingosine generation for sensing, not a sphingosine-supply pathway. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. |
| GO:0046512 sphingosine biosynthetic process | IDA PMID:9603946 Purification and characterization of a novel ceramidase from... | KEEP AS NON CORE | Summary: UniProt IDA: the enzyme generates sphingosine. Reason: True product of the hydrolysis; peripheral role. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. |
| GO:0046514 ceramide catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN004377481 for ceramide catabolic process. This is the reaction the enzyme performs. Reason: Ceramide hydrolysis is ceramide catabolism; core process. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. PMID:24465209 we generated a clean deletion in the neutral ceramidase encoded by PA0845 and measured enzyme activity from the PA5325-lacZ reporter construct pAL4. Ceramide fails to induce PA5325-lacZ in the absence of the neutral ceramidase |
| GO:0046514 ceramide catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro mapping to ceramide catabolic process. Reason: Consistent with the experimental role. Supporting Evidence: PMID:9603946 Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. |
| GO:0051872 sphingosine catabolic process | IDA PMID:24465209 Detection of host-derived sphingosine by Pseudomonas aerugin... | MODIFY | Summary: PseudoCAP IDA from PMID:24465209 (full text read). cerN generates sphingosine from ceramide; it is upstream of the sph genes that catabolise sphingosine and does not itself perform a catabolic step on sphingosine. Reason: Wrong term: the gene performs ceramide catabolism, producing sphingosine; it is not part of sphingosine catabolism. Re-pointed to GO:0046514 ceramide catabolic process (PSEUDOCAP WRONG_TERM, CONFIRMED). Proposed replacements: ceramide catabolic process Supporting Evidence: PMID:24465209 we generated a clean deletion in the neutral ceramidase encoded by PA0845 and measured enzyme activity from the PA5325-lacZ reporter construct pAL4. Ceramide fails to induce PA5325-lacZ in the absence of the neutral ceramidase |
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Download this section (compressed HTML)Q: Across the PTN004377481 neutral-ceramidase family, are the GO:0042759 long-chain fatty acid biosynthetic process and GO:0046512 sphingosine biosynthetic process annotations better expressed as ceramide catabolism with released products, or do mammalian members genuinely supply sphingosine as a signalling pathway?
Experiment: Test whether a cerN deletion alters P. aeruginosa fitness on ceramide as a carbon/sphingosine source and quantify sphingosine and free fatty acid release in vitro.
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