PutA (PA0782) is the bifunctional proline utilization A enzyme of Pseudomonas aeruginosa. It combines an N-terminal FAD-dependent proline dehydrogenase (PRODH) domain, which oxidises L-proline to 1-pyrroline-5-carboxylate (P5C) with quinone as the electron acceptor, and a C-terminal aldehyde dehydrogenase family P5C dehydrogenase (P5CDH) domain, which oxidises the P5C/L-glutamate 5-semialdehyde intermediate to L-glutamate with NAD+ (or, less well, NADP+). The two reactions together convert proline to glutamate, allowing proline to be used as a carbon and nitrogen source. Unlike enterobacterial PutA, the 1060-residue P. aeruginosa protein lacks the N-terminal ribbon-helix-helix DNA-binding domain and is not known to act as a transcriptional repressor; instead putA expression is activated by the AraC/XylS regulator PruR in response to proline. PutA is required for full virulence in a mouse acute pneumonia model and for oxidative-stress tolerance.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from IPR025703 (bifunctional PutA), a family that includes the enterobacterial PutAs, which carry an N-terminal ribbon-helix-helix DNA-binding domain and autoregulate putA/putP. The P. aeruginosa protein lacks that domain. Reason: P. aeruginosa PutA is 1060 residues with only PRODH and P5CDH domains, and putA is regulated by the separate AraC/XylS activator PruR. The mapping is over-propagated from RHH-containing PutAs and is wrong for this protein. Supporting Evidence: PMID:29632523 In P. aeruginosa, PutA is 1,060 amino acid residue long and contains only two domains, PRODH domain and P5CDH domain. PMID:29632523 It is unlikely that PutA functions as a transcriptional repressor to regulate the expression of Put system in P. aeruginosa. |
| GO:0003842 L-glutamate gamma-semialdehyde dehydrogenase (NAD+) activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the PTN000193023 IBD (5 seeds; PutA itself, E. coli and Salmonella PutA, S. aureus RocA and yeast Put2). PutA's own appearance in WITH/FROM is the expected self-seed. The node term is the shared P5CDH activity of bifunctional PutAs and monofunctional P5CDHs, so it suits the clade. Reason: Core activity of the C-terminal domain, directly assayed on the purified PAO1 enzyme; the IBD stands with or without this seed. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid |
| GO:0003842 L-glutamate gamma-semialdehyde dehydrogenase (NAD+) activity | IDA PMID:6819140 Purification and properties of the bifunctional proline dehy... | ACCEPT | Summary: PseudoCAP IDA and a seed of PTN000193023. Native PutA purified from PAO1 converts P5C to L-glutamate with NAD (Km 0.03 mM) or NADP (Km 0.17 mM). Reason: Seed verdict SOUND: purified-enzyme assay in the right organism, and NAD+ is the preferred cosubstrate, which matches the NAD+-specific term. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid PMID:6819140 Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP |
| GO:0003842 L-glutamate gamma-semialdehyde dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA for the P5CDH activity; consistent with the IDA. Reason: Same claim as the accepted IDA row. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid |
| GO:0004657 proline dehydrogenase activity | IDA PMID:6819140 Purification and properties of the bifunctional proline dehy... | ACCEPT | Summary: PseudoCAP IDA. The purified enzyme oxidises L-proline to P5C in an FAD-dependent reaction and is specific for the L-form. Reason: Direct assay of the purified PAO1 enzyme; core activity of the PRODH domain. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid |
| GO:0004657 proline dehydrogenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA for PRODH activity; consistent with the IDA. Reason: Same claim as the accepted IDA row. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO companion of the transcription factor row, from the same family-wide IPR025703 mapping. Reason: The autoregulatory repressor function belongs to RHH-containing enterobacterial PutAs; P. aeruginosa PutA lacks the domain and putA is controlled by PruR. Supporting Evidence: PMID:29632523 It is unlikely that PutA functions as a transcriptional repressor to regulate the expression of Put system in P. aeruginosa. |
| GO:0006562 L-proline catabolic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN000193023 (seeds fly P5CDH, yeast Put2, Salmonella PutA). PutA catalyses both steps of proline oxidation to glutamate. Reason: Direct participation (both catalytic steps). GO:0010133 proline catabolic process to glutamate is obsolete, so this is the most specific available term. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid |
| GO:0006562 L-proline catabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO for proline catabolism; consistent with the IBA and the enzymology. Reason: Same claim as the accepted IBA row. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid |
| GO:0009898 cytoplasmic side of plasma membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA seeded only by E. coli PutA, which shuttles to the inner membrane when proline is abundant. The PRODH domain reduces membrane quinone (EC 1.5.5.2), which makes a peripheral inner-membrane location plausible for P. aeruginosa PutA, but no localization study exists for this protein. Reason: Plausible from the quinone-dependent PRODH chemistry and the bifunctional PutA clade, but unverified for this protein and peripheral to the core activity. Supporting Evidence: file:PSEAE/putA/putA-uniprot.txt Reaction=L-proline + a quinone = (S)-1-pyrroline-5-carboxylate + a quinol + H(+) |
| GO:0009898 cytoplasmic side of plasma membrane | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule transfer of the same membrane location; see the IBA row. Reason: Same claim as the IBA row; plausible, unverified, non-core. Supporting Evidence: file:PSEAE/putA/putA-uniprot.txt Reaction=L-proline + a quinone = (S)-1-pyrroline-5-carboxylate + a quinol + H(+) |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Generic InterPro2GO oxidoreductase term. Reason: True but entailed by the accepted GO:0003842 and GO:0004657, both oxidoreductase activities. |
| GO:0055129 L-proline biosynthetic process | IEA GO_REF:0000120 | REMOVE | Summary: Electronic mapping that assigns the reverse (biosynthetic) direction of proline metabolism to a catabolic enzyme. Reason: PutA oxidises proline to glutamate; proline biosynthesis in bacteria runs through ProB, ProA and ProC, and the PutA reactions are not used in reverse. The IEA mapping is wrong for this enzyme. Supporting Evidence: PMID:6819140 Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid |
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Download this section (compressed HTML)Q: The PTN000193023 node carries a GO:0009898 cytoplasmic side of plasma membrane IBD seeded only by E. coli PutA, while yeast Put2 (mitochondrial) and fly P5CDH are seeds of other IBDs on the same node. Do eukaryotic leaves inherit 9898, or is it blocked by NOT annotations lower in the tree?
Q: Does P. aeruginosa PutA associate with the inner membrane in a proline-dependent way, as E. coli PutA does, despite lacking the RHH domain?
Experiment: Fractionate PAO1 grown with and without proline and immunoblot for PutA (with inner-membrane and cytoplasmic markers) to test membrane association.
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