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 a cytosolic 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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University of Nebraska Lincoln
Schools of Earth Sciences/Natur
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Christgen, Shelbi L; Zhu, Weidong; Sanyal, Nikhilesh et al. (2017) Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A. Biochemistry 56:6292-6303
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
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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
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
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
Zhang, Lu; Alfano, James R; Becker, Donald F (2015) Proline metabolism increases katG expression and oxidative stress resistance in Escherichia coli. J Bacteriol 197:431-40

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