Redesigned purification yields a fully functional PutA protein dimer from Escherichia coli.

Proline utilization by Escherichia coli and Salmonella typhimurium requires expression of genes putP (encoding a proline transporter) and putA. Genetic data indicate that the PutA protein is both put repressor and a respiratory chain-linked dehydrogenase. We report a redesigned purification procedure as well as the physical characteristics and biological activities of the PutA protein purified from E. coli. The purified protein was homogeneous as determined by electrophoresis performed under denaturing and nondenaturing conditions. Its N-terminal sequence corresponded to that predicted by the DNA sequence. We showed copurification of proline and delta 1-pyrroline-5-carboxylate dehydrogenase activities. Purified PutA protein bound put DNA in vitro in an electrophoretic band-shift assay and it could be reconstituted to inverted membrane vesicles, yielding proline dehydrogenase activity. The Stokes radius and Svedberg coefficient of the protein were determined to be 7.1 nm and 9.9 S, respectively. These hydrodynamic data revealed that the protein in our preparation was dimeric with a molecular mass of 293 kDa and that it had an irregular shape indicated by the friction factor (f/f0) of 1.6.     

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein.

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.     

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.     

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 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.     

Steady-state kinetic mechanism of the proline:ubiquinone oxidoreductase activity of proline utilization A (PutA) from Escherichia coli

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a β₈α₈-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest k_cat/K_m was obtained with CoQ₂. The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ₁, combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites.     

Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein

The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.