Bacterial sodium-coupled proline uptake and fused-PutA catabolism

A reusable bacterial module in which a PutP-family sodium/proline symporter imports L-proline and a fused PutA (proline utilization A) protein oxidizes it to L-glutamate in two catalytic steps. The FAD-dependent PutA proline dehydrogenase (PRODH) domain transfers electrons to a membrane quinone while forming 1-pyrroline-5-carboxylate (P5C), and after nonenzymatic ring opening the NAD-dependent glutamate-5-semialdehyde dehydrogenase (GSALDH) domain oxidizes glutamate 5-semialdehyde to L-glutamate. Fused PutA proteins occur mainly in Gram-negative bacteria. In lineages whose putA and putP genes are transcribed divergently, including Escherichia coli, Salmonella and Pseudomonas putida, PutA is trifunctional: an N-terminal ribbon-helix-helix (RHH) DNA-binding domain lets the same protein repress the put genes until proline accumulates. Other PutAs lack the RHH domain and have only the two catalytic activities.

Scope and generalization. The module is the generic bacterial PutP plus fused PutA proline-utilization system, not a P. putida-specific pathway. The experimental references are Salmonella typhimurium PutA (purification of the bifunctional enzyme, membrane association, DNA binding and autoregulation) and Escherichia coli PutA and PutP (kinetics, quinone coupling, reconstituted sodium/proline symport). P. putida KT2440 is kept as one exemplar because its put genes have direct pathway genetics. All three representative PutAs are trifunctional: KT2440 PutA also carries the N-terminal RHH DNA-binding domain (PMID:18767154) and represses its put genes. Transcriptional repression is excluded from the module because it is a separate, non-metabolic activity of the same protein, not a step in the proline-to-glutamate conversion. The RHH-less type A and type B PutAs (for example Bradyrhizobium japonicum BjPutA, PMID:28712849) show that the two catalytic steps occur without it. Taxon context stays at Bacteria because fused PutA proteins are also found outside the Gram-negative lineages that dominate the characterized set; lineages with separate PRODH and P5C dehydrogenase proteins (for example Bacillus PutB/PutC) are not instances of this module. The boundary begins with sodium-coupled proline import and ends with L-glutamate. PutA-dependent transcriptional repression, respiratory-chain reactions downstream of quinol, and proline biosynthesis are outside the module; the regulatory activity is recorded as a core function in the E. coli, Salmonella and P. putida PutA gene reviews. Grounding. The PutP step uses PANTHER PTHR48086:SF3 (SODIUM_PROLINE SYMPORTER), because the family as a whole is substrate-heterogeneous (PanF, ActP, DUR31) while SF3 is the PutP clade; both representatives are classified at SF3. The PRODH step uses InterPro IPR025703 (Bifunct_PutA), the full-length fused-PutA family, because PRODH activity lies outside the aldehyde-dehydrogenase domain. The GSALDH step uses NCBIfam TIGR01238, the PutA C-terminal-domain branch of P5C dehydrogenases (InterPro IPR005933). Both match E. coli, Salmonella and KT2440 PutA but not monofunctional B. subtilis RocA or yeast PUT2 (checked against the InterPro API). PANTHER is not used: PutA falls in PTHR42862:SF1, which also holds monofunctional fungal PUT2 and Firmicute RocA/PutC P5C dehydrogenases that lack the PRODH domain, so the PANTHER subfamily cannot select the fused architecture. The same PutA molecule appears in two leaves, one per active site. P5C ring opening to glutamate 5-semialdehyde is nonenzymatic and is represented in the connection between the two PutA reactions rather than as a protein-catalyzed part.