Electrochemical and Functional Characterization of the Proline Dehydrogenase Domain of the PutA Flavoprotein from Escherichia coli

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Abstract
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 Em (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 Em for PutA-bound FAD. The pH behavior of the Em 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.