The control of gene expression by enzymes provides a direct pathway for cells to respond to fluctuations in metabolites and nutrients. One example is the proline utilization A (PutA) protein from Escherichia coli. PutA is a membrane-associated enzyme that catalyzes the oxidation of L-proline to glutamate using a flavin containing proline dehydrogenase domain and a NAD+ dependent Δ1-pyrroline-5-carboxylate dehydrogenase domain. In some Gram-negative bacteria such as E. coli, PutA is also endowed with a ribbon-helix-helix DNA-binding domain and acts as a transcriptional repressor of the proline utilization genes. PutA switches between transcriptional repressor and enzymatic functions in response to proline availability. Molecular insights into the redox based mechanism of PutA functional switching from recent studies are reviewed. In addition, new results from cell-based transcription assays are presented which correlate PutA membrane localization with put gene expression levels. General membrane localization of PutA, however, is not sufficient to activate the put genes.
PutA; transcriptional regulation; membrane-binding; proline utilization; DNA-binding; multifunctional enzyme
Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane binding domain include major reorganization of the flavin ribityl chain, severe (35 degree) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5), Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-αl loop to complete the active site.
Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, l-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.
The first diffraction-quality crystals of a PutA protein are reported. One of the three crystal forms described here exhibits pseudo-merohedral twinning. Removal of the N-terminal histidine tag aided the crystallization of another form.
Proline utilization A proteins (PutAs) are large (1000–1300 residues) membrane-associated bifunctional flavoenzymes that catalyze the two-step oxidation of proline to glutamate by the sequential action of proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase domains. Here, the first successful crystallization efforts for a PutA protein are described. Three crystal forms of PutA from Bradyrhizobium japonicum are reported: apparent tetragonal, hexagonal and centered monoclinic. The apparent tetragonal and hexagonal crystals were grown in the presence of PEG 3350 and sodium formate near pH 7. The apparent tetragonal form diffracted to 2.7 Å resolution and exhibited pseudo-merohedral twinning such that the true space group is P212121 with four molecules in the asymmetric unit. The hexagonal form diffracted to 2.3 Å resolution and belonged to space group P6222 with one molecule in the asymmetric unit. Centered monoclinic crystals were grown in ammonium sulfate, diffracted to 2.3 Å resolution and had two molecules in the asymmetric unit. Removing the histidine tag was important in order to obtain the C2 crystal form.
proline utilization A; PutA; proline catabolism; proline dehydrogenase; P5C dehydrogenase; pseudo-merohedral twinning
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 Δ1-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 CoQ1 (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 CoQ1, a soluble analog 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 kcat during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental kcat and kcat/Km parameters.
Proline dehydrogenase (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. In Sinorhizobium meliloti, as in other microorganisms, the putA gene is transcriptionally activated in response to proline. In Rhodobacter capsulatus, Agrobacterium, and most probably in Bradyrhizobium, this activation is dependent on an Lrp-like protein encoded by the putR gene, located immediately upstream of putA. Interestingly, sequence and genetic analysis of the region upstream of the S. meliloti putA gene did not reveal such a putR locus or any other encoded transcriptional activator of putA. Furthermore, results obtained with an S. meliloti putA null mutation indicate the absence of any proline-responsive transcriptional activator and that PutA serves as an autogenous repressor. Therefore, the model of S. meliloti putA regulation completely diverges from that of its Rhizobiaceae relatives and resembles more that of enteric bacteria. However, some differences have been found with the latter model: (i) S. meliloti putA gene is not catabolite repressed, and (ii) the gene encoding for the major proline permease (putP) does not form part of an operon with the putA gene.
Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related monofunctional enzymes. Some of these extra features are predicted to be important for substrate channeling in BjPutA. Multiple sequence alignment analysis shows that some PutAs have a 17-residue conserved motif in the C-terminal 20–30 residues of the polypeptide chain. The BjPutA structure shows that this motif helps seal the internal substrate-channeling cavity from the bulk medium. Finally, it is shown that some PutAs have a 100–200 residue domain of unknown function in the C-terminus that is not found in minimalist PutAs. Remote homology detection suggests that this domain is homologous to the oligomerization beta-hairpin and Rossmann fold domain of BjPutA.
Proline Utilization A; PutA; Substrate Channeling; Proline Catabolism; Proline Metabolism; Proline Dehydrogenase; Pyrroline-5-Carboxylate Dehydrogenase; Pyrroline-5-Carboxylate; Glutamate Semialdehyde; Domain Repeat; Aldehyde Dehydrogenase; Flavoenzyme; Remote Homology Detection; Review
PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (Em) value of −0.132 V (pH 7.5) was determined for the bound FAD/FADH2 couple in BjPutA that is significantly more negative (∼ 55 mV) than the Em for EcPutA-bound FAD. The more negative Em value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated suggesting lipids influence the FAD redox environment. Accordingly, an Em value of −-0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val-402 in EcPutA is substituted at the analogous position in BjPutA with Ala-310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an Em value of −0.09 V (pH 7.5). The > 40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.
Proline utilization A (PutA) from Escherichia coli is a multifunctional flavoprotein that is both a transcriptional repressor of the proline utilization (put) genes and a membrane-associated enzyme which catalyzes the 4e- oxidation of proline to glutamate. Previously, proline was shown to induce PutA-membrane binding and alter the intracellular location and function of PutA. To distinguish the roles of substrate binding and FAD reduction in the mechanism of how PutA changes from a DNA-binding protein to a membrane-bound enzyme, the kinetic parameters of PutA-membrane binding were measured under different conditions using model lipid bilayers and surface plasmon resonance (SPR). The effects of proline, FAD reduction, and proline analogues on PutA-membrane associations were determined. Oxidized PutA shows no binding to E. coli polar lipid vesicles. In contrast, proline and sodium dithionite induce tight binding of PutA to the lipid bilayer with indistinguishable kinetic parameters and an estimated dissociation constant (KD) of < 0.01 nM (pH 7.4) for the reduced PutA-lipid complex. Proline analogues such as L-THFA and DL-P5C also stimulate PutA binding to E. coli polar lipid vesicles with KD values ranging from about 3.6 – 34 nM (pH 7.4) for the PutA-lipid complex. The greater PutA-membrane binding affinity (> 300-fold) generated by FAD reduction relative to the nonreducing ligands demonstrates that FAD reduction controls PutA-membrane associations. On the basis of SPR kinetic analysis with differently charged lipid bilayers, the driving force for PutA-membrane binding is primarily hydrophobic. In the SPR experiments membrane-bound PutA did not bind put control DNA confirming that the membrane-binding and DNA-binding activities of PutA are mutually exclusive. A model for the regulation of PutA is described in which the overall translocation of PutA from the cytoplasm to the membrane is driven by FAD reduction and the subsequent energy difference (∼ 24 kJ/mol) between PutA-membrane and PutA-DNA binding.
Proline dehydrogenase (PRODH) and Δ1-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 Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-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 to PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify 9 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 Km=27 mM and kcat=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 (KI=1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 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.
Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Δ1-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional E. 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 (Ki = 30 mM), L-lactate (Ki = 1 mM), and L- tetrahydro-2-furoic acid (L-THFA, Ki = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 Å. The discovery of acetate as a competitive inhibitor suggests the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen bonding and non-polar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a 5-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 Å 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 kcat 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.
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 kcat/Km 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.
PutA, proline metabolism; proline:ubiquinone oxidoreductase; proline dehydrogenase
PutA is a novel flavoprotein in Escherichia coli that switches from a transcriptional repressor to a membrane-bound proline catabolic enzyme. Previous crystallographic studies of the PutA proline dehydrogenase (PRODH) domain under oxidizing conditions revealed that the FAD N(5) and ribityl 2′- OH form hydrogen bonds with Arg431 and Arg556, respectively. Here we identify molecular interactions in the PutA PRODH active site that underlie redox-dependent functional switching of PutA. We report that reduction of the PRODH domain induces major structural changes in the FAD cofactor, including a 22° bend of the isoalloxazine ring along the N(5)-N(10) axis, crankshaft rotation of the upper part of the ribityl chain, and formation of a new hydrogen bond network involving the ribityl 2′- OH, FAD N(1) atom, and Gly435. The roles of the FAD 2′-OH group and the FAD N(5)-Arg431 hydrogen bond pair in regulating redox-dependent PutA-membrane associations were tested using FAD analogues and site-directed mutagenesis. Kinetic membrane-binding measurements and cell-based reporter gene assays of modified PutA proteins show that disrupting the FAD N(5)-Arg431 interaction impairs reductive activation of PutA-membrane binding. We also show that the FAD-2′-OH acts as a redox-sensitive toggle switch that controls PutA-membrane binding. These results illustrate a new versatility of the ribityl chain in flavoprotein mechanisms.
The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. Previously, the DNA binding activity of PutA, a 1320 amino acid polypeptide, was localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nM, which is slightly higher than that of the PutA-DNA complex (Kd ∼ 45 nM). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, exhibit in vivo transcriptional activity and bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nM for the PutA47-DNA complex. Thus, N-terminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors which are comprised of a two-stranded antiparallel β-sheet that recognizes DNA and a dimeric core of four α-helices.
The PutA protein from Salmonella typhimurium is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate, a reaction that is coupled to the transfer of electrons to the electron transport chain in the cytoplasmic membrane. The PutA protein is also a transcriptional repressor that regulates the expression of the put operon in response to the availability of proline. Despite extensive genetic and biochemical studies of the PutA protein, it was not known if the PutA protein carries out both of these two opposing functions while membrane associated or if instead it carries them out in different cellular compartments. To distinguish between these alternatives, we directly assayed the binding of purified PutA protein to DNA and membranes in vitro. The results indicate that wild-type PutA does not simultaneously associate with DNA and membranes. In addition, PutA superrepressor mutants that exhibit increased repression of the put genes show a direct correlation between decreased membrane binding and increased DNA binding. These results support a model in which the PutA protein shuttles between the membrane (where it acts as an enzyme but lacks access to DNA-binding sites) and the cytoplasm (where it binds DNA and acts as a transcriptional repressor), depending on the availability of proline.
Proline dehydrogenase (PRODH) catalyzes the oxidation of L-proline to Delta-1-pyrroline-5-carboxylate. PRODHs exhibit a pronounced preference for proline over hydroxyproline (trans-4-hydroxy-L-proline) as the substrate, but the basis for specificity is unknown. The goal of this study, therefore, is to gain insights into the structural determinants of substrate specificity of this class of enzyme, with a focus on understanding how PRODHs discriminate between the two closely related molecules, proline and hydroxyproline. Two site-directed mutants of the PRODH domain of Escherichia coli PutA were created: Y540A and Y540S. Kinetics measurements were performed with both mutants. Crystal structures of Y540S complexed with hydroxyproline, proline, and the proline analog L-tetrahydro-2-furoic acid were determined at resolutions of 1.75 Å, 1.90 Å and 1.85 Å. Mutation of Tyr540 increases the catalytic efficiency for hydroxyproline three-fold and decreases the specificity for proline by factors of twenty (Y540S) and fifty (Y540A). The structures show that removal of the large phenol side chain increases the volume of the substrate-binding pocket, allowing sufficient room for the 4-hydroxyl of hydroxyproline. Furthermore, the introduced serine residue participates in recognition of hydroxyproline by forming a hydrogen bond with the 4-hydroxyl. This result has implications for understanding substrate specificity of the related enzyme human hydroxyproline dehydrogenase, which has serine in place of tyrosine at this key active site position. The kinetic and structural results suggest that Tyr540 is an important determinant of specificity. Structurally, it serves as a negative filter for hydroxyproline by clashing with the 4-hydroxyl group of this potential substrate.
The PutA protein is a membrane-associated enzyme that catalyzes the degradation of proline to glutamate. Genetic evidence suggests that in the absence of proline, the PutA protein also represses transcription of the putA and putP genes. To directly determine whether PutA protein binds to the put control region, we analyzed gel retardation of put control region DNA by purified PutA protein in vitro. The put control region is 420 bp. Purified PutA protein bound specifically to several nonoverlapping fragments of control region DNA, indicating the presence of multiple binding sites in the control region. Electrophoretic abnormalities and behavior of circularly permuted fragments of control region DNA indicate that it contains a region of intrinsically curved DNA. To determine whether the multiple binding sites or the DNA curvature are important in vivo, two types of deletions were constructed: (i) deletions that removed sequences predicted to contribute to DNA curvature as well as potential operator sites and (ii) deletions that removed only potential operator sites. Both types of deletions increased expression of the put genes but were still induced by proline, indicating that multiple cis elements are involved in repression. These data suggest a model for put repression that invokes the formation of a complex between PutA protein molecules bound at different sites in the control region, brought into proximity by a loop of curved DNA.
Proline utilization A (PutA) is a membrane associated multifunctional enzyme that catalyzes the oxidation of proline to glutamate in a two step process. In certain Gram-negative bacteria such as Pseudomonas putida, PutA also acts as an auto repressor in the cytoplasm when an insufficient concentration of proline is available. Here the N-terminal residues 1–45 of PutA from P. putida (PpPutA45), are shown to be responsible for DNA binding and dimerization. The solution structure of PpPutA45 was determined using NMR methods, where the protein is shown to be a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. DNA sequence recognition by PpPutA45 was determined using DNA gel mobility shift assays and NMR chemical shift perturbations. PpPutA45 was shown to bind a 14 base-pair DNA oligomer (5′-GCGGTTGCACCTTT-3′). A model of the PpPutA45-DNA oligomer complex was generated using Haddock 2.1. The antiparallel β-sheet that results from PpPutA45 dimerization serves as the DNA recognition binding site by inserting into the DNA major groove. The dimeric core of four α-helices provides a structural scaffold for the β-sheet from which residues Thr5, Gly7, and Lys9 make sequence specific contacts with the DNA. The structural model implies flexibility of Lys9 which can either make hydrogen bond contacts with guanine or thymine. The high sequence and structure conservation of the PutA RHH domain suggest interdomain interactions play an important role in the evolution of the protein.
Pseudomonas putida; NMR solution structure; PutA; ribbon-helix-helix (RHH) structures; PutA-DNA complex
A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).
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 metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism.
Substrate Channeling; Proline Metabolism; Proline Dehydrogenase; PRODH; Pyrroline-5-carboxylate Dehydrogenase; P5CDH; Pyrroline-5-Carboxylate; P5C; Glutamic semialdehyde; GSA; Gamma-Glutamyl Kinase; Gamma-Glutamyl Phosphate Reductase; Pyrroline-5-Carboxylate Synthase; P5CS; Gamma-Glutamyl Phosphate; Review
The genes for proline utilization were fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mu d(Ap lac). Stable deletion derivatives of these fusions were selected and used to study the transcriptional regulation of the put genes. Analysis of these fusions showed that the putA gene product, a bifunctional oxidase-dehydrogenase, also serves to negatively control transcription of the putA and putP genes. Transcription of the put genes is repressed only in putA+ strains; this repression is lifted when exogenous proline is supplied. Transcription of the put genes is stimulated by cyclic AMP in putA+ and putA strains. Maximal induction of the put genes in putA+ strains requires oxygen or an alternative electron acceptor. This oxygen effect is mediated by the putA protein since putA mutants show maximal transcription even without an electron acceptor. The orientation of the Mu d(Ap lac) insertions was determined by formation of Hfr's via the lac homology on F'ts114 lac+. The direction of chromosome mobilization by these Mu d(Ap lac)-directed Hfr's demonstrated that the putP and putA genes are divergently transcribed from a central regulatory region lying between them.
The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of the proline utilization genes, while the human homologue of the PutA proline dehydrogenase (PRODH) domain plays critical roles in p53-mediated apoptosis and schizophrenia. We report the crystal structure of a 669-residue truncated form of PutA that exhibits both PRODH and DNA-binding activities, which represents the first structure of a PutA protein and the first structure of a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that found in the helix-turn-helix superfamily of DNA-binding proteins, and a beta/alpha barrel PRODH domain with bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.
The multifunctional Escherichia coli PutA flavoprotein functions as both a membrane-associated proline catabolic enzyme and transcriptional repressor of the proline utilization genes putA and putP. To better understand the mechanism of transcriptional regulation by PutA, we have mapped the put regulatory region, determined a crystal structure of the PutA ribbon-helix-helix domain (PutA52) complexed with DNA and examined the thermodynamics of DNA binding to PutA52. Five operator sites, each containing the sequence motif 5′-GTTGCA-3′, were identified using gel-shift analysis. Three of the sites are shown to be critical for repression of putA, whereas the two other sites are important for repression of putP. The 2.25 Å resolution crystal structure of PutA52 bound to one of the operators (operator 2, 21-bp) shows that the protein contacts a 9-bp fragment, corresponding to the GTTGCA consensus motif plus three flanking base pairs. Since the operator sequences differ in flanking bases, the structure implies that PutA may have different affinities for the five operators. This hypothesis was explored using isothermal titration calorimetry. The binding of PutA52 to operator 2 is exothermic with an enthalpy of −1.8 kcal/mol and a dissociation constant of 210 nM. Substitution of the flanking bases of operator 4 into operator 2 results in an unfavorable enthalpy of 0.2 kcal/mol and 15-fold lower affinity, which shows that base pairs outside of the consensus motif impact binding. The structural and thermodynamic data suggest that hydrogen bonds between Lys9 and bases adjacent to the GTTGCA motif contribute to transcriptional regulation by fine-tuning the affinity of PutA for put control operators.
proline utilization A; X-ray crystallography; isothermal titration calorimetry; ribbon-helix-helix; proline catabolism
L-Azetidine-2-carboxylate (AC) and 3,4-dehydro-D,L-proline (DHP) are toxic L-proline analogs that can be used to select bacterial mutants defective for L-proline transport. Mutants resistant to AC and DHP are defective for proline transport alone (putP mutants), and mutants resistant to AC but not to DHP are defective both in putP and in the closely linked proline dehydrogenase gene putA. Proline dehydrogenase oxidizes DHP but not AC, probably detoxifying the former compound. These observations were exploited in preparing an otherwise isogenic set of Escherichia coli K-12 strains with well-defined defects in the putP and putA genes. The results of this study suggest that the genetic and biochemical characteristics of proline utilization in E. coli K-12 are closely analogous to those of Salmonella typhimurium.