PutA is a novel flavoprotein in Escherichia coli that switches from a transcriptional repressor to a membrane-bound proline catabolic enzyme. Previous crystallographic studies of the PutA proline dehydrogenase (PRODH) domain under oxidizing conditions revealed that FAD N(5) and the ribityl 2'-OH group form hydrogen bonds with Arg431 and Arg556, respectively. Here we identify molecular interactions in the PutA PRODH active site that underlie redox-dependent functional switching of PutA. We report that reduction of the PRODH domain induces major structural changes in the FAD cofactor, including a 22 degrees bend of the isoalloxazine ring along the N(5)-N(10) axis, crankshaft rotation of the upper part of the ribityl chain, and formation of a new hydrogen bond network involving the ribityl 2'-OH group, FAD N(1), and Gly435. The roles of the FAD 2'-OH group and the FAD N(5)-Arg431 hydrogen bond pair in regulating redox-dependent PutA-membrane associations were tested using FAD analogues and site-directed mutagenesis. Kinetic membrane binding measurements and cell-based reporter gene assays of modified PutA proteins show that disrupting the FAD N(5)-Arg431 interaction impairs the reductive activation of PutA-membrane binding. We also show that the FAD 2'-OH group acts as a redox-sensitive toggle switch that controls PutA-membrane binding. These results illustrate a new versatility of the ribityl chain in flavoprotein mechanisms.

The multifunctional Escherichia coli proline utilization A (PutA) flavoprotein functions both as a membrane-associated proline catabolic enzyme and as a transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put-regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52, a polypeptide corresponding to residues 1-52 of E. coli PutA) complexed with DNA, and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gelshift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25-Å-resolution crystal structure of PutA52 bound to one of the operators (operator 2; 21 bp) shows that the protein contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic, with an enthalpy of − 1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and a 15-fold-lower affinity, showing that base pairs outside of the consensus motif impact binding. Structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.

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Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4e-oxidation of proline to glutamate. Previously, proline was shown to induce PutA-membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNAbinding protein to a membrane-bound enzyme, the kinetic parameters of PutA-membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA-membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (K D ) of < 0.01 nM (pH 7.4) for the reduced PutAlipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with K D values ranging from about 3.6 -34 nM (pH 7.4) for the PutA-lipid complex. The greater PutA-membrane binding affinity (> 300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA-membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA-membrane binding is primarily hydrophobic. In the SPR experiments membranebound PutA did not bind put control DNA confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼ 24 kJ/mol) between PutA-membrane and PutA-DNA binding.The multifunctional proline utilization A (PutA) flavoprotein from Escherichia coli and Salmonella typhimurium is both a transcriptional repressor and a membrane associated enzyme (1-5). PutA regulates the put regulon, which contains the genes putA and putP (Na + /proline transporter) that are transcribed in opposite directions from the put control intergenic DNA (1,5-7). PutA represses transcription of the put genes by binding to specific sequences in the put control DNA region (2,8). The enzyme action of PutA coordinates step-wise proline dehydrogenase (PRODH) and Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, allowing bacteria to utilize proline as a sole nitrogen and energy source. The PRODH active site couples the oxidation of proline to the reduction of noncovalently bound flavin adenine dinucleotide (FAD) to form Δ 1 -pyrroline-5-carboxylate (P5C) (9). Reduced FAD is subsequently oxidized by an electron acceptor in the membrane to regenerate oxidized FAD †