Progress 04/01/09 to 03/31/14
Outputs
Target Audience: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. Changes/Problems: Nothing significant to report during this reporting period. What opportunities for training and professional development has the project provided? This project provided training for undergraduate students (10), graduate students (6), postdoctoral fellows (1), and professional scientists (1). These trainees designed, performed, and analyzed the experiments and results. They also presented at group meetings, regional conferences, and national/international meetings. How have the results been disseminated to communities of interest? The results have been published or are in preparation to be published in the appropriate peer-reviewed journals in our field. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We reported kinetic insights into how the multifunctional PutA enzyme, which is responsible for proline catabolism in Escherichia coli, switches between DNA binding and membrane binding. We have shown that conformational changes induced by proline reduction of the flavin drive PutA-membrane binding and involve hydrogen bond rearrangements in the active site that propagate to the membrane binding domain. Understanding how flavin redox signals are ultimately transmitted to regulatory/functional domains is a novel area of research that has implications for how flavin binding domains contribute to diverse signaling pathways. We have also uncovered hysteresis in PutA substrate channeling and a previously unknown mechanism of activation. Understanding the mechanism of activation will benefit studies of other substrate channeling enzyme systems.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zhu W, Haile A, Smithen, D, Singh RK, Larson J, Tanner JJ, Becker DF. Involvement of the ?3-?3 loop of the Proline Dehydrogenase Domain in Allosteric Regulation of Membrane Association of Proline Utilization A. Biochemistry 2013, 52:4482-91. PMCID:PMC3731750
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liang X, Zhang L, Natarajan S, Becker DF. Proline mechanisms of stress survival. Antioxid. Redox Signal. 2013. 19:998-1011. PMCID:PMC3763223
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Moxley MA, Sanyal N, Krishnan N, Tanner JJ, Becker DF. Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and ?1-pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PUTA). J. Biol. Chem. 2014. [Epub ahead of print]. PMID:24352662
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Singh H, Arentson BW, Becker DF, Tanner JJ. Structures of the PutA Peripheral Membrane Flavoenzyme Reveal a Dynamic Substrate-Channeling Tunnel and the Quinone Binding Site. PNAS 2014. In press.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. Changes/Problems: Nothing significant to report during this reporting period. What opportunities for training and professional development has the project provided? This project provided research and educational training opportunities for undergraduate students (3), graduate students (5), postdoctoral fellows (1), and professional scientists (1). Students designed, performed, and analyzed the research results. They also presented at laboratory group meetings, local conferences, and at international meetings. How have the results been disseminated to communities of interest? The results have been published or are currently being prepared for publication to the appropriate peer-review journals in the field. What do you plan to do during the next reporting period to accomplish the goals? We are optimistic that we are close to achieving a full-length structure of a trifunctional PutA. Our plan for the next year is to test the diffraction of the new StPutA crystals, and if necessary, use surface mutagenesis to improve the crystallizability of StPutA. We also plan to perform site-directed mutagenesis of RcPutA to gain information about the spatial relationship between the CTD and the other domains of PutA. These experiments will help us fit our model of the CTD into the SAXS envelope of EcPutA, which will improve our working model of the structure of trifunctional PutAs. Mutation of the conserved residues Asp370 and Glu372 abrogates the ability of proline to induce functional membrane association. The crystal structures of the PRODH domain mutants PutA86-630D370N and PutA86-630D370A show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. From our results, we suggest that the b3-a3 loop may be involved in transmitting the status of the PRODH active site and flavin redox state to the distal membrane association domain. Future studies will work to build molecular links from the b3-a3 loop region to the membrane association domain of PutA. The results from Trp-Dansyl FRET assays strongly suggest that residues in the a-domain are part of the membrane association domain. Thus, our experiments over the next year will focus substantially on the α-domain as the major mediator of membrane association. We will complete the characterization of the site-directed Trp mutants and develop a model for the PutA-membrane binding interface.
Impacts
What was accomplished under these goals?
Strong progress was made during the last budget period. We performed the first characterization of the shape and oligomeric state of a long bifunctional PutA and obtained new crystals of a trifunctional PutA. We provided evidence that highly conserved Asp and Glu residues of the b3-a3 loop of the PRODH domain (ba)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. We have shown that removal of the carboxylate group of Asp370 or Glu372, or replacement of Asp370 with the nonionizable asparagine results in dysfunctional PutA membrane associations. Thus, it appears that the b3-a3 loop is involved in the signaling mechanism of PutA and suggests that conformational changes of Glu372 and Asp370 are part of the larger, global structural change that drives membrane binding. A manuscript describing these results has been submitted for publication in Biochemistry (manuscript ID: bi-2013-00338f). We also have new evidence that the membrane association domain may involve the α-domain. Understanding how flavin redox signals are ultimately transmitted to regulatory/functional domains is a novel area of research that has implications for how flavin binding domains contribute to diverse signaling pathways.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Luo M, Arentsen BW, Srivastava D, Becker DF, Tanner JJ. Crystal Structures and Kinetics of Monofunctional Proline Dehydrogenase Provide Insight into Substrate Recognition and Conformational Changes Associated With Flavin Reduction and Product Release. Biochemistry. 2012. 51:10099-10108. PMCID:PMC3525754
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Srivastava D, Singh RK, Moxley MA, Henzl MT, Becker DF, Tanner JJ. The Three- Dimensional Structural Basis of Type II Hyperprolinemia. J. Mol. Biol. 2012. 420:176-89. PMCID:PMC3372638
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding of the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was to obtain solution structural information on PutA from E. coli in collaboration with Dr. John Tanner at the University of Missouri-Columbia. His group performed shape reconstructions that showed PutA is a symmetric V-shaped dimer linked by the DNA binding domain. The model is consistent with a mechanism in which the interaction of PutA with the membrane prevents DNA-binding interactions. Thus, PutA membrane and DNA binding are mutually exclusive. The kinetic mechanism of the PRODH reaction including microscopic rate constants was determined using stopped-flow kinetics. An isomerization event (~ 2 s-1) after reduction of the flavin (~ 30 s-1) was observed in the stopped-flow absorbance traces. The isomerization step was interpreted as a conformational change that is necessary for PutA to switch from a soluble to a membrane bound enzyme. The rate limiting step in the overall catalytic mechanism was observed to be reoxidation of the flavin with ubiquinone (~ 6 s-1). All of the above findings were published and communicated at various scientific meetings and invited seminars. PARTICIPANTS: Mike Moxley, Ben Arentson, and Nikilish Sanyal, Graduate Research Assistants, Department of Biochemistry, University of Nebraska-Lincoln, Dr. Weidong Zhu, Postdoctoral fellow, University of Nebraska-Lincoln, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia . TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
PutA proteins from different bacteria are distinguished by the presence of a DNA binding domain. That is, trifunctional PutAs (e.g., E. coli PutA) exhibit DNA binding and enzymatic activity whereas bifunctional PutAs (e.g., PutA from Bradyrhizobium japonicum) lack a DNA binding domain and only have enzymatic activity. Surprisingly, the oligomeric structure of E. coli PutA (EcPutA) and B. japonicum PutA (BjPutA) are vastly different. BjPutA forms a dimer by interactions via an enzymatic domain whereas EcPutA forms a dimer via the DNA binding domain. The different oligomeric structure of EcPutA is likely related to the functional switching mechanism by which EcPutA shuttles between DNA and membrane binding. Our kinetic data provide the first rate constant for the conformational change in PutA that switches it from a DNA binding protein to a membrane bound enzyme. These results are providing key mechanistic details that will significantly aid our understanding of proline bioenergetics in bacteria and PutA functional switching.
Publications
- Moxley MA and Becker DF. Rapid Reaction Kinetics of the Proline Dehydrogenase in the Multifunctional Proline Utilization A Protein. Biochemistry 2012. 51:511-520. PMCID:PMC3254707
- Arentson BW, Sanyal N, Becker DF. Substrate channeling in proline metabolism. Front Biosci. 2012 17:375-388. PMCID:PMC3342669
- Moxley MA, Tanner JJ, and Becker DF. Steady-State Kinetic Mechanism of the Proline:Ubiquinone Oxidoreductase Activity of Proline Utilization A (PutA) from Escherichia coli. Arch. Biochem. Biophys. 2011. 516:113-120. PMCID: PMC322327513.
- Singh RK, Larson JD, Zhu W, Rambo RP, Hura GL, Becker DF, and Tanner JJ. Small-Angle X-ray Scattering Studies of the Oligomeric State and Quaternary Structure of the Trifunctional Proline Utilization A (PutA) Flavoprotein from Escherichia coli. J. Biol. Chem. 2011. 286: 43144-43153. PMCID: PMC3234867
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding of the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was to obtain solution structural information on PutA from E. coli in collaboration with Dr. John Tanner at the University of Missouri-Columbia. His group performed shape reconstructions that showed PutA is a symmetric V-shaped dimer linked by the DNA binding domain. The model is consistent with a mechanism in which the interaction of PutA with the membrane prevents DNA-binding interactions. Thus, PutA membrane and DNA binding are mutually exclusive. The kinetic mechanism of the PRODH reaction was investigated this year using a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ(1), 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. All of the above findings were published and/or communicated at various scientific meetings and invited seminars. PARTICIPANTS: Mike Moxley, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Dr. Weidong Zhu, Postdoctoral fellow, University of Nebraska-Lincoln, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
PutA proteins from different bacteria are distinguished by the presence of a DNA binding domain. That is, trifunctional PutAs (e.g., E. coli PutA) exhibit DNA binding and enzymatic activity whereas bifunctional PutAs (e.g., PutA from Bradyrhizobium japonicum) lack a DNA binding domain and only have enzymatic activity. Surprisingly, the oligomeric structure of E. coli PutA (EcPutA) and B. japonicum PutA (BjPutA) are vastly different. BjPutA forms a dimer by interactions via an enzymatic domain whereas EcPutA forms a dimer via the DNA binding domain. The different oligomeric structure of EcPutA is likely related to the functional switching mechanism by which EcPutA shuttles between DNA and membrane binding. Our kinetic data published this year 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. These results are providing key mechanistic details that will significantly aid our understanding of proline bioenergetics in bacteria and PutA functional switching.
Publications
- Becker DF, Zhu W, and Moxely MA. Flavin redox switching of protein functions. Antioxid. Redox Signal. 2011. 14:1079-91.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding of the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was to identify conformational changes in the active site that may be critical for redox regulation of PutA. Inactivation of PutA with the inhibitor N-propargyl glycine (PPG) generated a form of the enzyme that mimics proline reduced PutA. The x-ray crystal structure showed conformational changes of active site residues Arg431, Asp370 and two nearby Glu residues (E372 and E373) that comprise a glutamate cluster. We have characterized mutants D370A, D370N, and E373A. The kcat/Km values for D370A, D370N, and E373A are 6 M-1s-1, 25 M-1s-1, and 41 M-1s-1 (pH 8.0). These values are 2-10-fold lower than WT EcPutA (80 M-1s-1). The Em of the bound FAD in mutants D370A and D370N are about 25-mV more positive than WT EcPutA (Em = - 80 mV, pH 7.5) while the Em for E373A is similar to WT. Functional membrane association activity is diminished by 25-fold for D370A and D370N and by 3-fold for E373A relative to WT EcPutA. Cell-based assays show a significant loss of β-galactosidase activity (D370A) or a loss of response to proline (D370N, E373A). These results support a key role for D370 in communicating with the flavin and possibly mediating signals out of the flavin active site. We have characterized the mechanism of ubiquinone (CoQ) reduction by EcPutA by steady-state and rapid reaction kinetics using various quinone analogs and inhibitors. Our results suggest a two-site ping-pong mechanism for EcPutA proline:ubiquinone oxidoreductase activity. We have also shown a ping-pong mechanism for the PRODH enzyme from yeast. All of the above findings were published and/or communicated at various scientific meetings and invited seminars. PARTICIPANTS: Mike Moxley, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Dr. Weidong Zhu, Postdoctoral fellow, University of Nebraska-Lincoln, Dr. Javier Seravalli, Department of Biochemistry, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia. TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The combined structural and biochemical information gained from PPG inactivated PutA provide evidence that redox changes in the flavin are transmitted via Arg431 and Asp370 to the glutamate cluster which leads out of the active site. These redox initiated conformational changes are then postulated to result in activation of PutA-membrane binding. Our kinetic studies of ubiquinone reduction by PutA have helped us conclude that ubiquinone and proline most likely have different binding sites. These results are providing key mechanistic details that will significantly aid our understanding of proline bioenergetics in bacteria and PutA functional switching.
Publications
- Wanduragala S, Sanyal N, Liang X, and Becker DF. 2010. Purification and Characterization of Put1p from Saccharomyces cerevisiae. Arch. Biochem. Biophys. 498:136-42.
- Srivastava D, Schuermann JP, White TA, Krishnan N, Sanyal N, Hura GL, Tan A, Henzl MT, Becker DF, Tanner JJ. 2010. Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum. PNAS. 107:2878-83.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding of the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was to identify conformational changes in the active site that may be critical for redox regulation of PutA. Inactivation of PutA with the inhibitor N-propargyl glycine (PPG) resulted in a three carbon covalent link between the N5 atom of the flavin and Lys329 in the active site. Characterization of the inactivated PutA enzyme revealed it behaved similar to proline reduced PutA. Global conformational changes detected by limited proteolysis and lipid binding studies provided evidence that PPG inactivated PutA mimics the proline reduced form of PutA. Subsequently, the structure of PPG inactivated PutA was solved. The x-ray crystal structure showed conformational changes of active site residues. Namely, Arg431, Asp370 and three nearby Glu residues that comprise a glutamate cluster. Site-directed mutagenesis is now in progress to test the roles of Asp370 and the glutamate cluster in the reductive activation mechanism of PutA membrane binding. Another key output this year was the kinetic characterization of the oxidative half-reaction of PutA. Using different ubiquinone analogs we showed that catalytic turnover of proline by PutA follows a ping pong mechanism. Binding of proline or an inhibitor molecule to PutA does not decrease oxidation rates of the flavin by ubiquinone, indicating that ubiquinone binds to a second site on the enzyme. Presently, we are exploring two potential ubiquinone binding sites that are within electron transfer distance from the flavin. All of the above findings were submitted for publication and were also communicated at various scientific meetings and invited seminars. PARTICIPANTS: Ashley Haile, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Mike Moxley, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Dr. Weidong Zhu, Postdoctoral fellow, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PARTICIPANTS: Ashley Haile, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Mike Moxley, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Dr. Weidong Zhu, Postdoctoral fellow, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The combined structural and biochemical information gained from PPG inactivated PutA provide evidence that redox changes in the flavin are transmitted via Arg431 and Asp370 to the glutamate cluster which leads out of the active site. These redox initiated conformational changes are then postulated to result in activation of PutA-membrane binding. Our kinetic studies of the PutA oxidative half-reaction have helped us conclude that ubiquinone is the physiological electron acceptor and that ubiquinone occupies a different binding site than proline. Most likely proline reduced flavin is oxidized by two successive one electron transfer steps to ubiquinone. These results are providing key mechanistic details that will significantly aid our understanding of PutA membrane interactions which are critical for regulating and catalyzing proline utilization in bacteria.
Publications
- Halouska S, Zhou Y, Becker DF, and Powers RP. Solution structure of Pseudomonas putida protein PpPutA45 and its DNA complex. Proteins 2009. 75(1):12-27.
- Srivastava D, Zhu W, Johnson Jr WH, Whitman CP, Becker DF, and Tanner JJ.Structure of the PutA PRODH Domain Inactivated by N-propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction. Biochemistry 2009, in revision.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding for the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was to identify a consensus sequence (GTTGCA) for PutA binding. The put-regulatory region, a 419-bp region that separates the putA and putP genes, contains five GTTGCA PutA binding motifs or operators. We elucidated the roles of these operators in repressing the expression of putA and putP by cell-based expression assays. A x-ray crystal structure by Dr. John Tanner's group of PutA52 bound to one of the operators shows that PutA contacts a 9-bp fragment corresponding to the GTTGCA consensus motif plus three flanking base pairs. Based on the arrangement of the five PutA-DNA binding sites, PutA most likely represses the put genes by hindering the σ70-dependent binding of E. coli RNA polymerase to the putA and putP promoters. The mechanism by which PutA switches between DNA-binding and membrane-bound enzymatic activity is redox dependent. We have shown that proline-dependent localization of wild-type PutA on the membrane correlates with the activation of lacZ reporter gene expression in cell based assays. From this work, we built a model for the regulation of PutA in which reduction of the FAD cofactor and subsequent PutA-membrane binding activate expression of the put genes, illustrating how gene expression can be controlled by sequestering a regulator on the membrane. Secondary structure analysis of PutA indicated that a membrane binding domain may be contained in C-terminal residues 1295-130. A deletion construct of these residues in PutA (PutA1294) eliminated PutA-membrane localization. PutA1294 behaves as a super-repressor in cell-based lacZ reporter gene assays demonstrating that PutA-membrane localization is critical to relieve autorepression of the putA gene. We have found though that PutA can still act as a repressor when artificially localized to the membrane which supports the model that protein-membrane localization in general is not sufficient to suppress regulatory proteins from binding to chromosomal DNA in bacteria. All of the above findings were published and were also communicated at various scientific meetings and invited seminars. PARTICIPANTS: Navasona Krishnan, Graduate Research Assistant, Department of Biochemistry-University of Nebraska-Lincoln, Yuzhen Zhou, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Derrick Anderson, Undergraduate Student, University of Nebraska-Lincoln, Ashley Haile, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Mike Moxley, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln, Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have gained considerable molecular details on how PutA represses transcription of the put genes. Evaluation of put control DNA sequences from other bacteria suggests that the GTTGCA motif is the fundamental transcriptional control element of the PutA autogenous repression system in Gram negative bacteria. We have also demonstrated that the flavin redox state alone governs the conformation and membrane-binding activity of PutA. The reduced PutA conformer exhibits enhanced membrane binding that sequesters PutA on the membrane, thus leading to the activation of the put genes. Because PutA can still act as a repressor when artificially localized to the membrane, specific conformational changes that coincide with membrane localization are needed to prevent protein-DNA binding. Our results provide an example of a unique mechanism by which a FAD cofactor controls membrane binding of a large multifunctional enzyme and illustrates a new versatility of flavins in transcription regulation.
Publications
- Halouska S, Zhou Y, Becker DF, and Powers RP. Solution structure of Pseudomonas putida protein PpPutA45 and its DNA complex. Proteins 2008. Epub ahead of print.
- Zhou Y, Larson JD, Bottoms CA, Arturo EC, Jenkins JL, Nix JC, Henzl MT, Becker DF, and Tanner JJ. Molecular and Structural Basis of Transcriptional Regulation of the put Regulon in Escherichia coli. J. Mol. Biol. 2008. 381:174-188.
- Krishnan N, Doster A, Duhamel G, and Becker DF. Characterization of a Helicobacter hepaticus putA mutant strain in host colonization and oxidative stress. Infect. Immun. 2008. 76:3037-3044.
- Zhou Y, Zhu W, Bellur PS, Rewinkel D, and Becker DF. Direct linking of metabolism and gene expression in the proline utilization A protein from Escherichia coli. Amino Acids 2008. 35:711-718.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The overall goal of this study is to provide molecular and structural understanding for the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane associated flavoenzyme. In certain prokaryotes such as Escherchia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization genes putA and putp (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment from our work this past year was determining the mechanistic roles key residues in the PRODH domain. 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. We showed that the N(5)-Arg431 interaction is critical for reductive activation of PutA-membrane binding and that the FAD-2'-OH acts as a redox-sensitive toggle switch that controls PutA-membrane binding. Structural data also support a unique role for the FAD-2'-OH group. Reduction of the PRODH domain in PutA was observed to induce major structural changes in the FAD cofactor, including a 22 degree 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. Another important advance this year was determining the sites at which PutA binds to the central regulatory DNA region between the putP and putA genes, which are transcribed in opposite directions from different promoters. We mapped the regulatory region by
site-mutational analysis and cell-based transcription reporter assays. Five operator sites each containing the 5'-GTTGCA-3' sequence motif were identified. Three sites are critical for repression of putA, while two other sites are important for repression of putP. This work has led to structural analysis of PutA-DNA interactions. All of the above findings were published or are presently under review. Our results were also communicated at various scientific meetings.
PARTICIPANTS: Navasona Krishnan, Graduate Research Assistant, Department of Biochemistry-University of Nebraska-Lincoln Yuzhen Zhou, Graduate Research Assistant, Department of Biochemistry, University of Nebraska-Lincoln Derrick Anderson, Undergraduate Student, University of Nebraska-Lincoln Prof. John J. Tanner, Collaborator, Departments of Chemistry and Biochemistry, University of Missouri-Columbia
TARGET AUDIENCES: Our project is providing summer research opportunities for undergraduate students from smaller institutions and from underrepresented groups.
PROJECT MODIFICATIONS: No significant changes to report.
Impacts
Our results illustrate a new versatility of the ribityl chain 2'-OH group in flavoprotein mechanisms. The structural changes observed in the PRODH domain of PutA suggest a mechanism for how redox signals are transmitted out of the flavin binding site. We have also gained key insights into how PutA represses transcription of the putA and putP genes. Our work has facilitated the knowledge of the structural basis of PutA-DNA interactions. A 22-bp oligonucleotide was used to generate a co-crystal structure of the PutA-DNA complex. We originally mapped the consensus sequence of PutA-DNA binding to a 6 bp fragment GTTGCA. The structure shows that the protein contacts a 9-bp fragment, corresponding to the consensus motif plus three flanking base pairs. It is thought that variations in the flanking pairs generate differences in PutA binding affinity to the different sites. This work has been submitted for publication and is currently under review.
Publications
- Krishnan N, Dickman MB, and Becker DF. 2007. Proline Modulates the Intracellular Redox Environment and Protects Mammalian Cells Against Oxidative Stress. Free Rad. Biol. Med. in press.
- White TA, Krishnan N, Becker DF, and Tanner JJ. 2007. Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus. J. Biol. Chem. 2007. 282: 14316-14327.
- Zhang, W, Zhang, M, Zhu, W, Wanduragula, S, Rewinkel, D, Tanner, JJ, and Becker, DF. 2007. Redox-Induced Changes in Flavin Structure and Roles of Flavin N(5) and the Ribityl 2'-OH in Regulating PutA-membrane Binding. Biochemistry. 46:483-91.
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Progress 10/01/05 to 09/30/06
Outputs
The overall goal of this study is to provide molecular and structural understanding for the redox-based functional switching of PutA, a multifunctional enzyme involved in regulating and catalyzing proline metabolism. The two-step conversion of proline to glutamate in Gram-negative bacteria is catalyzed by PutA (proline utilization A), a large membrane-associated flavoenzyme. In certain prokaryotes such as Escherichia coli, PutA also contains a ribbon-helix-helix (RHH) DNA binding domain and is an autogenous transcriptional repressor of the proline utilization (put) genes putA and putP (encodes a high affinity proline transporter). A major focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. A key accomplishment during the last year was the characterization of a bifunctional PutA homologue from B. japonicum (BjPutA) that lacks the RHH domain of EcPutA. BjPutA has allowed us
to compare the membrane-binding properties between bifunctional and trifunctional PutAs. Surface plasmon resonance (SPR) analysis of BjPutA lipid-binding revealed that both the oxidized and proline-reduced forms of BjPutA bind the membrane which contrasts with the highly proline-dependent membrane-binding behavior of EcPutA. From the results of this work, we propose that EcPutA has at least two membrane binding domains, one which is conserved in both bifunctional and trifunctional PutAs and a second domain which is unique to trifunctional PutAs. We have also demonstrated that BjPutA-lipid associations are entropically driven but are also significantly influenced by electrostatic effects with binding favored to negatively charged lipids. One residue that can contribute both electrostatic and hydrophobic interactions is arginine which has a positively charged guanidinium group that can interact with the negatively charged phosphate groups and an aliphatic chain that can localize in the
hydrophobic region of the membrane bilayers. Indeed, examination of the BjPutA and EcPutA proline dehydrogenase domain structures show a conserved solvent-exposed Arg residue (R330 and R425 in BjPutA and EcPutA, respectively) located in a loop region near the proline dehydrogenase domain. Structurally, it is reasonable that lipid binding to this region would facilitate electron transfer from the FAD cofactor to a membrane electron acceptor during turnover. Preliminary data with EcPutA, suggest that the unique membrane binding domain of trifunctional PutAs is in the C-terminal region. We have also confirmed the proposed RHH domain in EcPutA by X-ray crystallography of a truncated PutA protein containing residues 1-52 (EcPutA52). The DNA recognition element of the RHH fold is an anti-parallel beta-sheet that forms in the dimer complex and binds to the major groove of the DNA. We have shown that Lys9 of the beta-sheet is critical for EcPutA-DNA binding. The RHH dimer is stabilized by
intersubunit helix-helix hydrophobic interactions and significantly stabilizes the dimeric structure of full-length EcPutA.
Impacts
Knowledge gained from this study will contribute to the understanding of redox sensitive regulatory proteins, metabolism-linked transcriptional regulation and how gene expression is modulated by subcellular localization of transcriptional regulators. Also, this work will provide insights into how multifunctional proteins coordinate different activities in response to cellular conditions. Control of gene expression through protein-membrane interactions is relevant to a broad range of organisms including plants and animals.
Publications
- Chen, C, Wanduragula, S, Becker, DF, Dickman, MD. 2006. A Tomato QM-like Protein Protects Saccharomyces cerevisiae Cells Against Oxidative Stress by Regulating Intracellular Proline Levels. Appl. Environ. Microbiol. 72:4001-4006.
- Larson, JD, Schuermann, JP, Zhou, Y, Jenkins, JL, Becker, DF, and Tanner, JJ. 2006. Crystal Structures of the DNA-binding Domain of Escherichia coli Proline utilization A Flavoprotein and Analysis of the Role of Lys9 in DNA Recognition. Protein Sci. 15:1-12.
- Zhang, W, Becker, DF, Cheng, Q. 2006. A Mini-Review of Recent W.O. Patents (2004-2005) of Novel Anti-fungal Compounds in the Field of Anti-infective Drug Targets. Recent Patents on Anti-Infective Drug Discovery 1 (2):225-230.
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Progress 10/01/04 to 09/30/05
Outputs
Proline utilization A (PutA) is a membrane-associated enzyme in gram negative bacteria that converts proline to glutamate using separate flavin-dependent proline dehydrogenase (PRODH) and NAD-dependent delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. In some bacteria such as Escherichia coli, PutA contains a ribbon-helix-helix (RHH) DNA-binding domain and is a transcriptional repressor of the proline utilization (put) genes putA (encodes PutA) and putP (encodes the high-affinity proline transporter PutP). Thus, PutA from E. coli (EcPutA) is a remarkable trifunctional enzyme that transforms from a gene regulatory protein into a membrane-bound enzyme. Our focus is to understand how trifunctional PutA proteins integrate catalytic, membrane binding and DNA binding activities within a single polypeptide. We have extended our understanding of redox-dependent conformational changes in EcPutA by investigating the intrinsic Trp fluorescence properties of a
truncated EcPutA protein and discovered that W211 is the main molecular marker of the fluorescence quenching induced by proline. An apparent rate constant of 0.6 s-1 was determined for the proline-dependent fluorescence decrease by stopped-flow fluorescence measurements of PutA86-630. This indicates that the switching of EcPutA from a transcriptional repressor to a membrane-bound enzyme occurs on a time scale that is much slower than the limiting rate constant for proline reduction of the flavin cofactor in PutA (133 s-1), consistent with flavin reduction preceding proline-induced conformational changes. We also characterized PutA from the Bradyrhizobium japonicum (BjPutA), which lacks DNA binding activity and is a bifunctional PutA homologue. Using spectroelectrochemistry, we discovered that the flavin in BjPutA has a redox potential that is 55-mV more negative that EcPutA. This results in only partial reduction of the flavin cofactor in BjPutA by proline. Subsequently, we identified
a residue in the flavin binding site of EcPutA that helps tune the redox potential of the flavin cofactor so that proline fully reduces the flavin. This residue (Val402) is conserved in all trifunctional PutAs enabling efficient regulation by proline. Examination of the membrane binding properties of BjPutA demonstrated that the tight regulation of PutA-membrane associations by proline reduction of the flavin cofactor occurs only in trifunctional PutAs. Last, we characterized PutA homologues from Helicobacter species and observed that unlike EcPutA and BjPutA, Helicobacter PutA has significant oxygen reactivity during turnover with proline which may influence redox homeostasis in certain ecological niches.
Impacts
Knowledge gained from this study will contribute to the understanding of redox sensitive regulatory proteins, metabolism-linked transcriptional regulation and how gene expression is modulated by subcellular localization of transcriptional regulators. Also, this work will provide insights into how multifunctional proteins coordinate different activities in response to cellular conditions. Control of gene expression through protein-membrane interactions is relevant to a broad range of organisms including plants and animals.
Publications
- Krishnan, N. and Becker, D. F. Characterization of a Bifunctional PutA Homologue from Bradyrhizobium japonicum and Identification of an Active Site Residue that Modulates the Redox Potential of the Flavin Adenine Dinucleotide Cofactor. Biochemistry, 2005. 44: 9130-9139.
- Zhu W., Becker DF. Exploring the Proline-Dependent Conformational Change in the Multifunctional PutA Flavoprotein by Tryptophan Fluorescence Spectroscopy. Biochemistry, 2005. 44: 12297-306.
- Zhang, W., Krishnan, N. and Becker DF. Kinetic and Thermodynamic Analysis of Bradyrhizobium japonicum PutA-membrane Associations. Arch. Biochem. Biophys., 2005. in press.
- Krishnan, N., and Becker, DF. Oxygen Reactivity of PutA from Helicobacter Species and Proline-linked Oxidative Stress. J. Bacteriol., 2005. in press.
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Progress 10/01/03 to 09/30/04
Outputs
We identified and characterized the DNA binding domain in PutA and showed PutA is a member of the ribbon-helix-helix (RHH) family of transcriptional repressors. We also demonstrated that flavin reduction induces PutA-membrane binding. Using surface plasmon resonance, we showed oxidized PutA does not bind the membrane, in contrast, a tight PutA-membrane complex is formed upon flavin reduction. From this work we described a thermodynamic model for the regulation of PutA in which the expression of the put genes is activated by reduction of the flavin and subsequent binding of PutA to the membrane.
Impacts
Our findings provide a model for how gene expression can be controlled by sequestering a transcriptional regulator on the membrane. Control of gene expression through protein-membrane interactions is relevant to a broad range of organisms including plants and animals.
Publications
- Gu, D., Zhou, Y., Kallhoff, V., Baban, B., Tanner, J. J., and Becker, D. F. 2004. Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme. J. Biol. Chem. 279: 31171-31176.
- Baban, B. A., Vinod, M. P., Tanner, J. J., and Becker, D. F. 2004 Probing a Hydrogen Bond Pair and the FAD Redox Properties in the Proline Dehydrogenase Domain of Escherichia coli PutA. Biochim. Biophys. Acta 1701:49-59.
- Zhang, M., White, T. A., Schuermann, J. P., Baban, B. A., Becker, D. F. and Tanner, J. J. 2004 Structures of the Escherichia coli PutA Proline Dehydrogenase Domain in Complex with Competitive Inhibitors. Biochemistry 43:12539-12548.
- Zhang, W., Zhou, Y., and Becker, D. F. 2004. Regulation of PutA-Membrane Associations by FAD Reduction. Biochemistry, 43:13165-13174.
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