Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli

The multifunctional PutA flavoprotein from Escherichia coli is a peripherally membrane-bound enzyme that has both proline dehydrogenase (PDH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. In addition to its enzymatic functions, PutA displays DNA-binding activity and represses proline catabolism by binding to the control region DNA of the put regulon (put intergenic DNA). Presently, information on structure -function relationships for PutA is derived from primary structure analysis. To gain further insight into the functional organization of PutA, our objective is to dissect PutA into different domains and to characterize them separately. Here, we report the characterization of a bifunctional proline dehydrogenase (PutA₆₆₉) that contains residues 1-669 of the PutA protein. PutA₆₆₉ purifies as a dimer and has a PDH specific activity that is 4-fold higher than that of PutA. As anticipated, PutA₆₆₉ lacks P5CDH activity. At pH 7.5, an E_m (E-FAD/E-FADH^-) of -0.091 V for the two-electron reduction of PutA₆₆₉-bound FAD was determined by potentiometric titrations, which is 15 mV more negative than the E_m for PutA-bound FAD. The pH behavior of the E_m for PutA₆₆₉-bound FAD was measured in the pH range 6.5-9.0 at 25 °C and exhibited a 0.03 V/pH unit slope. Analysis of the DNA and membrane-binding properties of PutA₆₆₉ shows that it binds specifically to the put intergenic control DNA with a binding affinity similar to that of PutA. In contrast, we did not observe functional association of PutA₆₆₉ with membrane vesicles. We conclude that PutA₆₆₉ has FAD-binding and DNA-binding properties comparable to those of PutA but lacks a membrane-binding domain necessary for stable association with the membrane.