Redox Modulation of Oligomeric State in Proline Utilization A

Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family.     

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.     

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.     

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.     

Catabolic pathway of arginine in Anabaena involves a novel bifunctional enzyme that produces proline from arginine

Arginine participates widely in metabolic processes. The heterocyst‐forming cyanobacterium Anabaena catabolizes arginine to produce proline and glutamate, with concomitant release of ammonium, as major products. Analysis of mutant Anabaena strains showed that this catabolic pathway is the product of two genes, agrE (alr4995) and putA (alr0540). The predicted PutA protein is a conventional, bifunctional proline oxidase that produces glutamate from proline. In contrast, AgrE is a hitherto unrecognized enzyme that contains both an N‐terminal α/β propeller domain and a unique C‐terminal domain of previously unidentified function. In vitro analysis of the proteins expressed in Escherichia coli or Anabaena showed arginine dihydrolase activity of the N‐terminal domain and ornithine cyclodeaminase activity of the C‐terminal domain, overall producing proline from arginine. In the diazotrophic filaments of Anabaena, β‐aspartyl‐arginine dipeptide is transferred from the heterocysts to the vegetative cells, where it is cleaved producing aspartate and arginine. Both agrE and putA were found to be expressed at higher levels in vegetative cells than in heterocysts, implying that arginine is catabolized by the AgrE‐PutA pathway mainly in the vegetative cells. Expression in Anabaena of a homolog of the C‐terminal domain of AgrE obtained from Methanococcus maripaludis enabled us to identify an archaeal ornithine cyclodeaminase.     

Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress

Proline is converted to glutamate in two successive steps by the proline utilization A (PutA) flavoenzyme in gram-negative bacteria. PutA contains a proline dehydrogenase domain that catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidation of proline to Δ¹-pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase domain that catalyzes the NAD⁺-dependent oxidation of P5C to glutamate. Here, we characterize PutA from Helicobacter hepaticus (PutA_Hh) and Helicobacter pylori (PutA_Hp) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutA_Hh and PutA_Hp lack DNA binding activity, in contrast to PutA from Escherichia coli (PutA_Ec), which both regulates and catalyzes proline utilization. PutA_Hh and PutA_Hp display catalytic activities similar to that of PutA_Ec but have higher oxygen reactivity. PutA_Hh and PutA_Hp exhibit 100-fold-higher turnover numbers (~30 min⁻¹) than PutA_Ec (<0. 3 min⁻¹) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutA_Hh forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutA_Hh and PutA_Hp was probed by oxidative stress studies in E. coli. Expression of PutA_Ec and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutA_Hp and PutA_Hh expression dramatically reduces E. coli cell survival and is correlated with relatively lower proline levels and increased hydrogen peroxide formation. The discovery of reduced oxygen species formation by PutA suggests that proline catabolism may influence redox homeostasis in the ecological niches of these Helicobacter species.