Redox Properties of the PutA Protein from Escherichia coli and the Influence of the Flavin Redox State on PutA−DNA Interactions

Abstract

The PutA flavoprotein from Escherichia coli is both a transcriptional repressor and a membrane-associated proline dehydrogenase. PutA represses transcription of the putA and putP genes by binding to the control region DNA of the put regulon (put intergenic DNA). Previous work has shown that FAD has a role in regulating the transcriptional repressor and membrane binding functions of the PutA protein. To test the influence of the FAD redox state on PutA--DNA interactions, we characterized the redox properties of the PutA flavoprotein from E. coli. At pH 7.5, an E(m)(E--FAD/E--FADH(2)) of --0.076 V for the two-electron reduction of PutA-bound FAD was determined by potentiometric titrations. Stabilization of semiquinone species was not observed during potentiometric measurements. Dithionite reduction of PutA, however, caused formation of red anionic semiquinone. The E(m) value for the proline/Delta(1)-pyrroline-5-carboxylate couple was determined to be --0.123 V, demonstrating the reduction of PutA by proline is favored by a potential difference (Delta E degrees ') of more than 0.045 V. Characterization of the PutA redox properties in the presence of put intergenic DNA revealed an E(m)(E(DNA)--FAD/E(DNA)--FADH(2)) of --0.086 V. The 10 mV negative shift in E(m) corresponds to just a 2.3-fold increase in the dissociation constant of PutA with the DNA upon reduction of FAD. Thus, it appears the FAD redox state has little influence on the overall PutA--DNA interactions.

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

“…Sequence-specific synthetic oligonucleotides were purchased from Integrated DNA Technologies. The put intergenic DNA (419 bp) used for DNA binding assays was prepared as described previously using genomic DNA from E. coli strain JT31 (14).…”

“…NdeI digestion of the modified putA gene resulted in PutA⌬85. PutA47 (residues 1-47) and PutA90 (residues 1-90) constructs were engineered by positioning a BamHI site at amino acid codons 48 and 91, respectively, of the putA gene in pET3a (14). BamHI digestion then removed the corresponding PutA amino acid codons 48 -1320 in PutA47 and codons 91-1320 in PutA90.…”

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

“…PRODH was then partially purified by Ni-NTA chromatography using previously described methods and stored at 4 °C in potassium phosphate buffer containing 100 mM KCl, 10% glycerol, and 10% sucrose [28]. PRODH activity was detected using the proline:dichloroindophenol (DCPIP) oxidoreductase assay as previously described [29]. Proline:O 2 activity was determined by following the reduction of cytochrome c at 550 nm and the production of P5C in air-saturated 20 mM MOPS (pH 7.5) buffer as previously described [30,31].…”

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

“…For example, K m value of proline for the ProDH domains of PutA enzymes in P. aeruginosa [6], S. typhimurium [7] and P. fluorescence [3] has been reported 45, 43, and 35 mM respectively. As it has been noted in the literatures, high K m value of ProDHs for proline is one of the common features of proline metabolizing enzymes in bacteria [11]. Therefore, the higher substrate affinity of P. putida ProDH toward proline made this enzyme very attractive for use in biosensors.…”

“…ProDH activity was spectrophotometrically assayed using proline as substrate, INT (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride) oxidoreductase assay which was performed by INT as a terminal electron acceptor and Phenazine methosulfate (PMS) as a mediator electron carrier [11]. Assay reaction mixture (final volume, 1.0 ml) contained 100 mM Tris-HCl, 10 mM MgCl 2 , 10 % glycerol, pH 8.5, 200 mM L-proline, 0.2 mM FAD, 0.45 mM INT, 0.08 mM PMS and the enzyme.…”