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

Biochemistry. 2002 May 21;41(20):6525-32. doi: 10.1021/bi025706f.


The multifunctional PutA flavoprotein from Escherichia coli is a peripherally membrane-bound enzyme that has both proline dehydrogenase (PDH) and Delta(1)-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 (PutA(669)) that contains residues 1-669 of the PutA protein. PutA(669) purifies as a dimer and has a PDH specific activity that is 4-fold higher than that of PutA. As anticipated, PutA(669) lacks P5CDH activity. At pH 7.5, an E(m) (E-FAD/E-FADH(-)) of -0.091 V for the two-electron reduction of PutA(669)-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 PutA(669)-bound FAD was measured in the pH range 6.5-9.0 at 25 degrees C and exhibited a 0.03 V/pH unit slope. Analysis of the DNA and membrane-binding properties of PutA(669) 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 PutA(669) with membrane vesicles. We conclude that PutA(669) 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Flavoproteins / chemistry*
  • Flavoproteins / genetics
  • Macromolecular Substances
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification
  • Peptide Fragments / chemistry*
  • Peptide Fragments / genetics
  • Peptide Fragments / isolation & purification
  • Potentiometry / methods
  • Proline Oxidase / chemistry*
  • Proline Oxidase / genetics
  • Proline Oxidase / isolation & purification
  • Protein Binding / genetics
  • Protein Structure, Tertiary / genetics
  • Structure-Activity Relationship


  • Bacterial Proteins
  • Flavoproteins
  • Macromolecular Substances
  • Membrane Proteins
  • Multienzyme Complexes
  • Peptide Fragments
  • PutA protein, Bacteria
  • Proline Oxidase