putA encodes a large fused proline-utilization protein with FAD-dependent proline dehydrogenase and NAD-dependent glutamate-5-semialdehyde dehydrogenase domains. It converts L-proline to L-glutamate in two coupled oxidative steps and also represses the divergently transcribed putA and putP promoters.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000976 transcription cis-regulatory region binding | IEA GO_REF:0000117 | ACCEPT | Summary: The N-terminal PutA RHH domain binds the conserved put control DNA sequence. Reason: Direct structural and binding experiments with the P. putida PutA N-terminal domain establish sequence-specific binding to put control DNA. Supporting Evidence: file:PSEPK/putA/putA-uniprot.txt DR InterPro; IPR048798; PutA_RHH. PMID:18767154 PpPutA45 was shown to bind a 14 base-pair DNA oligomer PMID:18767154 DNA sequence recognition by PpPutA45 was determined using DNA gel mobility shift assays |
| GO:0001217 DNA-binding transcription repressor activity | IEA GO_REF:0000117 | ACCEPT | Summary: DNA-binding transcription repressor activity is directly supported for P. putida PutA. Reason: putA knockout causes constitutive expression of both put promoters, while a catalytically inactive PutA retains regulation. Supporting Evidence: PMID:11097893 The PutA protein acts as a repressor of put gene expression in P. putida PMID:11097893 This regulatory activity is independent of the catabolic activity of PutA |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | MODIFY | Summary: The generic transcription-factor term is sound but less informative than the supported repressor term. Reason: Direct P. putida evidence establishes repression rather than an unspecified transcription-factor activity. Proposed replacements: DNA-binding transcription repressor activity Supporting Evidence: PMID:11097893 The PutA protein acts as a repressor of put gene expression in P. putida |
| GO:0003842 L-glutamate gamma-semialdehyde dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: The NAD-dependent second oxidation step is a core PutA activity. Reason: The exact UniProt record assigns EC 1.2.1.88 and RHEA:30235, while sequence homology in the P. putida study assigns the corresponding C-terminal domain. Supporting Evidence: file:PSEPK/putA/putA-uniprot.txt Reaction=L-glutamate 5-semialdehyde + NAD(+) + H2O = L-glutamate + NADH PMID:10613867 both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide |
| GO:0004657 proline dehydrogenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: FAD-dependent proline dehydrogenase is the first core PutA activity. Reason: The exact record assigns EC 1.5.5.2 and RHEA:23784. P. putida genetics links PutA to proline utilization, while sequence homology assigns the proline-dehydrogenase region within the fused protein. Supporting Evidence: file:PSEPK/putA/putA-uniprot.txt Reaction=L-proline + a quinone = (S)-1-pyrroline-5-carboxylate PMID:10613867 both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MODIFY | Summary: The generic regulation term is correct but less informative than the supported negative-regulation child. Reason: Promoter-expression experiments establish repression, which is captured specifically by GO:0045892. Proposed replacements: negative regulation of DNA-templated transcription Supporting Evidence: PMID:11097893 expression from the put promoters is constitutive in a host background with a knockout putA gene |
| GO:0006562 L-proline catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: PutA performs both oxidative steps of L-proline catabolism to glutamate. Reason: A putA insertion prevents growth on proline as sole carbon and nitrogen source, and the fused protein carries both catalytic domains. Supporting Evidence: PMID:10613867 unable to grow with proline as the sole carbon and nitrogen source PMID:10613867 both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide file:PSEPK/putA/putA-deep-research-openscientist.md PutA thus enables *P. putida* to use proline as a **carbon, nitrogen, and energy source.** |
| GO:0009898 cytoplasmic side of plasma membrane | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: The membrane-facing location is mechanistically plausible but is not the sole state of this regulatory enzyme. Reason: Type C PutA proteins switch between soluble DNA-bound and peripheral membrane-associated states; the exact KT2440 location has not been directly measured in the cited studies. Supporting Evidence: PMID:28712849 flavin redox dependent changes in subcellular location and function in response to proline |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: This parent term is true but obscures the two known substrate-specific activities. Reason: GO:0004657 and GO:0003842 separately capture both catalytic activities with exact reaction support. Proposed replacements: proline dehydrogenase activity L-glutamate gamma-semialdehyde dehydrogenase activity Supporting Evidence: file:PSEPK/putA/putA-uniprot.txt EC=1.5.5.2 file:PSEPK/putA/putA-uniprot.txt EC=1.2.1.88 |
| GO:0043168 anion binding | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Generic anion binding does not describe a distinct PutA function. Reason: This ARBA parent-level binding prediction is uninformative beside the two specific catalytic activities and lacks direct KT2440 binding evidence. Supporting Evidence: file:PSEPK/putA/putA-goa.tsv GO:0043168 anion binding |
| GO:0045892 negative regulation of DNA-templated transcription | IEA GO_REF:0000108 | ACCEPT | Summary: Negative transcriptional regulation is experimentally established for PutA. Reason: Loss of putA causes constitutive expression from both divergent put promoters. Supporting Evidence: PMID:11097893 The PutA protein acts as a repressor of put gene expression in P. putida |
| GO:0055129 L-proline biosynthetic process | IEA GO_REF:0000120 | REMOVE | Summary: The biosynthesis annotation is a directionally incorrect propagation from the shared semialdehyde-dehydrogenase domain. Reason: Direct P. putida evidence and both exact reactions place PutA in oxidative proline catabolism to glutamate, not glutamate-to-proline biosynthesis. Supporting Evidence: PMID:10613867 both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide file:PSEPK/putA/putA-uniprot.txt glutamate; L-glutamate from L-proline: step 1/2. |
| GO:0098562 cytoplasmic side of membrane | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: This broader membrane-side annotation is plausible but secondary and redundant with GO:0009898. Reason: PutA membrane association is conditional in the characterized regulatory class, and the exact KT2440 subcellular switch remains inferred. Supporting Evidence: PMID:28712849 flavin redox dependent changes in subcellular location and function in response to proline |
| GO:1901363 heterocyclic compound binding | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: Generic heterocycle binding is not an informative standalone PutA function. Reason: Binding of FAD, NAD, and proline is already inherent in the specific catalytic activities; no separate generic binding role is established. Supporting Evidence: file:PSEPK/putA/putA-goa.tsv GO:1901363 heterocyclic compound binding |
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Download this section (compressed HTML)Q: Does KT2440 PutA undergo the same directly measurable FAD-redox-dependent membrane switch as E. coli PutA?
Q: What is the identity of the sigma-54-dependent positive regulator required for full put promoter induction?
Experiment: Measure coupled proline-to-glutamate turnover and intermediate channeling with purified Q88D80 and catalytic-domain mutants.
Type: coupled enzyme kinetics
Experiment: Compare membrane association and operator binding of oxidized and proline-reduced Q88D80.
Type: membrane-binding and DNA-binding assay
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