Proline utilization A (PutA) from Escherichia coli is a large multifunctional protein that uniquely combines enzymatic and transcriptional regulatory activities within a single polypeptide. As an enzyme, PutA peripherally associates with the inner cytoplasmic membrane to catalyze the four-electron oxidation of proline to glutamate via the coordinated actions of separate flavin-dependent proline dehydrogenase (PRODH) and NAD-dependent A1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. An N-terminal ribbon-helix-helix motif endows PutA with DMA-binding activity enabling PutA to function also as acytosolic autogenous transcriptional represser of the proline utilization (put)genes putA and putP (encodes a high affinity proline transporter). To fulfill its mutually exclusive functions as a transcriptional represser and membrane-bound proline catabolic enzyme, PutA undergoes proline-dependent functional switching. The central hypothesis of this proposal is that flavin redox signals generated in the PRODH active site control the global conformation, subcellular location and function of PutA. This idea is supported by recent work demonstrating that reduction of the flavin cofactor drives PutA-membrane association and induces structural changes in the PRODH active site. To further explore this hypothesis, a dynamic and structural model for how the flavin cofactor controls functional switching of PutA will be developed using a wide variety of approaches, including spectroelectrochemistry, site-directed mutagenesis, surface plasmon resonance, X- ray crystallography, hydrophobic photolabeling and hydrogen-deuterium exchange mass spectrometry. The major goals of this study are to uncover the novel redox-based mechanism whereby PutA transforms from a gene regulatory protein into a membrane-bound enzyme and to provide a structural understanding of how PutA integrates catalytic, membrane-binding and DMA-binding activities within a single polypeptide.
The specific aims to achieve this are the following: 1. Identify flavin-protein interactions that direct the functional switching of PutA. 2. Elucidate the global three-dimensional architecture of trifunctional PutA. 3. Identify membrane-binding domains of PutA. 4. Characterize proline-dependent conformational changes in PutA. This work will generate mechanistic insights into how proteins perform multiple tasks. Project outcomes will also further the understanding of proline bioenergetics in gastric cancer, trypanosomal diseases, type I hyperprolinemia and schizophrenia susceptibility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM061068-06A1S1
Application #
7393932
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ikeda, Richard A
Project Start
2000-01-01
Project End
2011-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
6
Fiscal Year
2007
Total Cost
$41,628
Indirect Cost
Name
University of Nebraska Lincoln
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
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Tanner, John J; Fendt, Sarah-Maria; Becker, Donald F (2018) The Proline Cycle As a Potential Cancer Therapy Target. Biochemistry 57:3433-3444
Korasick, David A; Gamage, Thameesha T; Christgen, Shelbi et al. (2017) Structure and characterization of a class 3B proline utilization A: Ligand-induced dimerization and importance of the C-terminal domain for catalysis. J Biol Chem 292:9652-9665
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Korasick, David A; Pemberton, Travis A; Arentson, Benjamin W et al. (2017) Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline. Molecules 23:
Liu, Li-Kai; Becker, Donald F; Tanner, John J (2017) Structure, function, and mechanism of proline utilization A (PutA). Arch Biochem Biophys 632:142-157
Moxley, Michael A; Zhang, Lu; Christgen, Shelbi et al. (2017) Identification of a Conserved Histidine As Being Critical for the Catalytic Mechanism and Functional Switching of the Multifunctional Proline Utilization A Protein. Biochemistry 56:3078-3088
Arentson, Benjamin W; Hayes, Erin L; Zhu, Weidong et al. (2016) Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA. Biosci Rep 36:
Tanner, John J (2016) Empirical power laws for the radii of gyration of protein oligomers. Acta Crystallogr D Struct Biol 72:1119-1129
Luo, Min; Gamage, Thameesha T; Arentson, Benjamin W et al. (2016) Structures of Proline Utilization A (PutA) Reveal the Fold and Functions of the Aldehyde Dehydrogenase Superfamily Domain of Unknown Function. J Biol Chem 291:24065-24075

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