PhaZ is the intracellular medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase of Pseudomonas putida KT2440. It is associated with PHA granules and mobilizes stored polyester during carbon-storage turnover, with knockout strains accumulating higher and more persistent PHA levels. The KT2440 locus is PP_5004 (UniProt Q88D24); UniProt currently exposes the primary gene name as phaB, but the protein description, ESTHER family assignment, and Pseudomonas literature identify the gene product as PhaZ.
Definition: Catalysis of the hydrolysis of ester bonds in medium-chain-length poly(3-hydroxyalkanoate), yielding 3-hydroxyalkanoate monomers and oligomers.
Justification: GO:0050527 names poly(3-hydroxyoctanoate), whereas PhaZ hydrolyzes mcl-PHAs containing multiple aliphatic and aromatic monomers. A chemistry-appropriate broader mcl-PHA depolymerase term is needed to represent this substrate scope.
Parent term: hydrolase activity
Supporting Evidence:
Definition: The chemical reactions and pathways resulting in the breakdown of poly(3-hydroxyalkanoate), an intracellular carbon- and energy-storage polyester.
Justification: GO has the specific molecular-function term GO:0050527 and the broader process term GO:0042620, but it lacks a direct catabolic-process term for intracellular PHA mobilization by depolymerases such as PhaZ.
Parent term: poly(3-hydroxyalkanoate) metabolic process
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004806 triacylglycerol lipase activity | IEA GO_REF:0000118 | MODIFY | Summary: This automated TreeGrafter annotation assigns a generic lipase substrate class that does not match the biology of PP_5004. The protein is curated in UniProt as a poly(3-hydroxyalkanoate) depolymerase, and the relevant GO term already exists as GO:0050527. Reason: Replace the triacylglycerol-specific activity term with the dedicated PHA depolymerase term. PhaZ mobilizes intracellular mcl-PHA granules, not glycerolipids. Proposed replacements: poly(3-hydroxyoctanoate) depolymerase activity Supporting Evidence: PMID:17170116 We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. file:PSEPK/phaZ/phaZ-uniprot.txt SubName: Full=Poly(3-hydroxyalkanoate) depolymerase file:PSEPK/phaZ/phaZ-notes.md The best current molecular-function term is GO:0050527 poly(3-hydroxyoctanoate) depolymerase activity rather than triacylglycerol lipase activity. file:PSEPK/phaZ/phaZ-deep-research-falcon.md acting **almost exclusively on mcl-PHAs** |
| GO:0046503 glycerolipid catabolic process | IEA GO_REF:0000118 | MODIFY | Summary: This process term is misleading for phaZ. PhaZ acts in intracellular PHA mobilization at PHA granules, whereas glycerolipid catabolism implies a different substrate class and cellular context. Reason: GO:0042620 poly(3-hydroxyalkanoate) metabolic process is the closest existing biological-process term. A more specific catabolic child term would be preferable, but the current glycerolipid annotation should not be kept. Proposed replacements: poly(3-hydroxyalkanoate) metabolic process Supporting Evidence: PMID:17170116 We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. file:PSEPK/phaZ/phaZ-notes.md GO:0046503 glycerolipid catabolic process is misleading because intracellular PHA mobilization is not glycerolipid catabolism; GO:0042620 poly(3-hydroxyalkanoate) metabolic process is closer, but GO currently lacks a direct poly(3-hydroxyalkanoate) catabolic process term for intracellular PHA mobilization. file:PSEPK/phaZ/phaZ-deep-research-falcon.md PP_5004 functions in **PHA mobilization**, coupling polymer breakdown to carbon and energy homeostasis |
| GO:0070088 polyhydroxyalkanoate granule | IDA PMID:17170116 Biochemical evidence that phaZ gene encodes a specific intra... | NEW | Summary: PhaZ is a granule-associated intracellular depolymerase rather than a soluble glycerolipid catabolic enzyme. GO has a dedicated cellular component term for the PHA granule, which is the relevant site of action. Reason: Granule association is central to PhaZ function and is more informative than generic cytoplasmic localization. Supporting Evidence: PMID:17170116 We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. file:PSEPK/phaZ/phaZ-notes.md PhaZ is associated with polyhydroxyalkanoate granules rather than glycerolipid catabolism. file:PSEPK/phaZ/phaZ-deep-research-falcon.md Evidence places PhaZ as an **intracellular, granule-associated** enzyme acting on the surface of PHA inclusions rather than a secreted extracellular depolymerase |
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Download this section (compressed HTML)Q: Which of the additional putative PhaZ paralogs in the KT2440 genome are active depolymerases, and do they target distinct PHA chemistries or growth states?
Q: What nutritional or regulatory signals control phaZ expression and granule recruitment during transitions from PHA accumulation to mobilization?
Experiment: Measure PHA amount and monomer composition over time in wild type, clean Delta phaZ, and complemented PP_5004 strains during nitrogen starvation and refeeding on octanoate or lignin-derived substrates.
Hypothesis: Loss of phaZ blocks intracellular PHA mobilization and prolongs high polymer loads after accumulation.
Type: genetic perturbation plus polymer quantification
Experiment: Localize epitope-tagged or antibody-detected PhaZ across growth phases by fluorescence microscopy or immunogold electron microscopy and correlate localization with granule morphology and turnover.
Hypothesis: PhaZ is recruited to PHA granules specifically during mobilization phases.
Type: localization assay
Experiment: Test purified PP_5004 against mcl-PHA latex, defined hydroxyalkanoate oligomers, triacylglycerols, and p-nitrophenyl esters to refine substrate specificity for GO curation.
Hypothesis: PP_5004 prefers intracellular mcl-PHA substrates and does not behave as a generic triacylglycerol lipase.
Type: biochemical enzyme assay
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