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.     

Structure and kinetics of monofunctional proline dehydrogenase from Thermus thermophilus

Proline dehydrogenase (PRODH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-A resolution structure of Thermus thermophilus PRODH reveals a distorted (betaalpha)(8) barrel catalytic core domain and a hydrophobic alpha-helical domain located above the carboxyl-terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-A shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is -75 mV and the kinetic parameters for proline are K(m) = 27 mm and k(cat) = 13 s(-1). 3,4-Dehydro-l-proline was found to be an efficient substrate, and l-tetrahydro-2-furoic acid is a competitive inhibitor (K(I) = 1.0 mm). Finally, we demonstrate that T. thermophilus PRODH reacts with O(2) producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.     

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.     

Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA

The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide. Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment. To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612). Disruption of the D370-Y540 pair only slightly diminished k(cat), while more noticeable affects were observed in K(m). The mutant D370A/Y540F showed the most significant changes in the pH dependence of k(cat)/K(m) and K(m) relative to wild-type PutA86-601 with an apparent pK(a) value of about 8.2 for the pH-dependent decrease in K(m). From the pH profile of D370A/Y540F inhibition by l-tetrahydro-2-furoic acid (l-THFA), the pH dependency of K(m) in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex. Replacement of D370 and Y540 produces divergent effects on the E(m) for bound FAD. At pH 7.0, E(m) values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively. The 40-mV positive shift in E(m) determined for D370A relative to wild-type PutA86-601 (E(m)=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment.     

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.     

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.     

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.     

Structure-activity characterization of sulfide:quinone oxidoreductase variants

Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane protein that catalyzes the oxidation of sulfide species to elemental sulfur. The enzymatic reaction proceeds in two steps. The electrons from sulfides are transferred first to the enzyme cofactor, FAD, which, in turn, passes them onto the quinone pool in the membrane. Several wild-type SQR structures have been reported recently. However, the enzymatic mechanism of SQR has not been fully delineated. In order to understand the role of the catalytically essential residues in the enzymatic mechanism of SQR we produced a number of variants of the conserved residues in the catalytic site including the cysteine triad of SQR from the acidophilic, chemolithotrophic bacterium Acidithiobacillus ferrooxidans. These were structurally characterized and their activities for each reaction step were determined. In addition, the crystal structures of the wild-type SQR with sodium selenide and gold(I) cyanide have been determined. Previously we proposed a mechanism for the reduction of sulfides to elemental sulfur involving nucleophilic attack of Cys356 on C(4A) atom of FAD. Here we also consider an alternative anionic radical mechanism by direct electron transfer from Cys356 to the isoalloxazine ring of FAD.     

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ¹-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ¹-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD⁺-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.     

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.