The membrane-associated form of PutA does not bind put promoter DNA; thus, the enzymatic and DNA-binding activities of PutA are mutually exclusive [7]. The proline dehydrogenase (PRODH) activity resides in the amino-terminal domain following the DNA-binding domain; a truncated protein containing residues 1-669 retains proline dehydrogenase and DNA-binding activity, but lacks membrane association and Δ¹-pyrroline-5-carboxylate dehydrogenase activity [8]. Proline dehydrogenase activity requires the presence of an electron acceptor. The reaction is split into a reductive half reaction (reduction of the FAD cofactor by oxidation of proline) and an oxidative half reaction (reoxidation of reduced |FRAME: FADH2| by transfer of electrons to the quinone pool in the cytoplasmic membrane) [9]. The proline:ubiquinone oxidoreductase reaction proceeds via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites [10]. Stopped-flow kinetic analysis of both half reactions indicate that reoxidation of the flavin cofactor is rate-limiting [11]. The conserved H487 residue is critical for reversibility of the PRODH reaction as well as the kinetics of the conformational change and functional switching to the transcription factor activity [12]. Association of the enzyme with the membrane is dependent on reduction of the FAD cofactor, which induces a change in the conformation of the protein [13, 14, 15]. Both proline binding and FAD reduction contribute to the conformational change [16, 17]. The β3-α3 loop of the proline dehydrogenase (β-α)₈ barrel may transmit the proline-mediated allosteric signal that affects membrane association of PutA [18]. A conserved C-terminal motif comprising residues 1300-1320 together with the α domain (residues 142-259) appears to form the membrane-binding domain of PutA [19]. Crystal structures of the amino-terminal proline dehydrogenase domain have been reported [17, 20, 21, 22, 23], and site-directed mutants in various active site residues have been characterized [17, 21, 22, 24]. These studies resulted in a detailed picture of the mechanism of the redox-dependent structural changes of the protein and identified Y540 as an important substrate specificity determinant. A model of the entire protein including small-angle X-ray scattering (SAXS) data shows that PutA is a symmetric V-shaped dimer. Parts of the C-terminal domain may function as a lid that covers the internal substrate-channeling cavity [5]. The C-terminal domain shows similarity to aldehyde dehydrogenases, indicating that the true substrate for the second enzymatic activity may be γ-glutamamate semialdehyde, which is thought to spontaneously equilibrate with Δ¹-pyrroline-5-carboxylate (P5C) [25]. Steady-state and transient kinetic data for the individual dehydrogenase reactions as well as the coupled PRODH-Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) reaction have been obtained. The P5CDH reaction data was fitted to an ordered ternary reaction mechanism [26]. For the coupled PRODH-P5CDH reaction, the substrate channeling step is rate limiting; in addition, it shows a ~20-fold slower k_cat when measured during single-turnover compared to steady state, suggesting activation of the channeling step [26]. Expression of putA is reduced by prolonged exposure to osmotic stress [27]. A putA mutant is more sensitive to oxidative stress than wild type. Proline metabolism produces H₂O₂, which induces the OxyR regulon, and thereby katG expression [28]. putA, among other genes involved in carbon source transport and metabolism, was downregulated in two MG1655 lysogens carrying closely related Stx2a phages O104 and PA8 1208335|. PutA: "proline utilization A" [29] Reviews: [30, 31, 32, 33, 34, 35, 36, 37, 38]