The overall goal of this study is to provide molecular and structural understanding for the redox based functional switching of 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. PutA catalyzes the four-electron oxidation of proline to glutamate by coordinating the activities of separate flavin-dependent proline dehydrogenase (PRODH) and NAD+-dependent 1-pyrroline- 5-carboxylate dehydrogenase (P5CDH) domains. 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 genes putA and putP (encodes a high affinity proline transporter). To fulfill its mutually exclusiv functions as a transcriptional repressor and membrane-bound enzyme, PutA undergoes proline-dependent functional switching. Thus, PutAs with DNA binding activity are unique trifunctional flavoproteins that act as sensors of cellular metabolism by responding to proline availability. Earlier studies have established that proline reduction of the flavin activates PutA membrane- binding thereby triggering PutA switching from a transcriptional repressor to a membrane-bound enzyme. The principal hypothesis of this proposal is that redox signals in the flavin active site control the conformation, subcellular localization, and function of PutA. The goal of this study is to further examine this hypothesis by building a structural and dynamic model for how reduction of the flavin cofactor drives PutA functional switching. Several major milestones achieved in the previous funding period form the basis for the proposed studies. In particular, conformational changes in the flavin itself and surrounding active site residues were identified and shown to be critical for initiating functional switching. The thermodynamic and structural basis of the PutA repressor function was elucidated. The first crystal structure of a full-length bifunctional PutA was determined. The solution structure of a trifunctional PutA was modeled using SAXS data and crystal structures of domains. And most recently, the elusive membrane-binding domain of PutA was identified. These results provide an outstanding framework for understanding, at unprecedented detail, the molecular mechanisms whereby PutA transforms from a gene regulatory protein to a membrane-bound enzyme. A new direction integrated into this study is to understand how proline catabolism is coupled to reduction of the respiratory chain in vivo.
The specific aims are the following: 1. Determine the organization and structure of trifunctional PutA. 2. Characterize the bioenergetics of proline metabolism. 3. Elucidate the mechanism of functional switching in PutA.

Public Health Relevance

The amino acid proline has multifaceted roles that impact human health. Inborn errors in proline metabolic genes are manifested in neurological dysfunctions such as schizophrenia, increased incidence of seizures, connective tissue diseases, premature aging, and osteopenia. This project will further the understanding of proline metabolism in cancer preventing mechanisms, type I hyperprolinemia, schizophrenia susceptibility, and pathogens that rely on proline as a major fuel source, such as the causative agents of peptic ulcers (Helicobacter pylori) and African sleeping sickness (Trypanosoma brucei).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061068-12
Application #
8668068
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Barski, Oleg
Project Start
2001-08-01
Project End
2016-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
12
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Nebraska Lincoln
Department
Biochemistry
Type
Earth Sciences/Resources
DUNS #
City
Lincoln
State
NE
Country
United States
Zip Code
68583
Tanner, John J (2016) Empirical power laws for the radii of gyration of protein oligomers. Acta Crystallogr D Struct Biol 72:1119-1129
Arentson, Benjamin W; Hayes, Erin L; Zhu, Weidong et al. (2016) Engineering a Trifunctional Proline Utilization A Chimera By Fusing a DNA-Binding Domain to a Bifunctional PutA. Biosci Rep :
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
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
Spencer, Andrea L M; Bagai, Ireena; Becker, Donald F et al. (2014) Protein/protein interactions in the mammalian heme degradation pathway: heme oxygenase-2, cytochrome P450 reductase, and biliverdin reductase. J Biol Chem 289:29836-58
Singh, Harkewal; Arentson, Benjamin W; Becker, Donald F et al. (2014) Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site. Proc Natl Acad Sci U S A 111:3389-94
Luo, Min; Christgen, Shelbi; Sanyal, Nikhilesh et al. (2014) Evidence that the C-terminal domain of a type B PutA protein contributes to aldehyde dehydrogenase activity and substrate channeling. Biochemistry 53:5661-73
Zhu, Weidong; Haile, Ashley M; Singh, Ranjan K et al. (2013) Involvement of the *3-*3 loop of the proline dehydrogenase domain in allosteric regulation of membrane association of proline utilization A. Biochemistry 52:4482-91
Moxley, Michael A; Becker, Donald F (2012) Rapid reaction kinetics of proline dehydrogenase in the multifunctional proline utilization A protein. Biochemistry 51:511-20
Singh, Ranjan K; Tanner, John J (2012) Unique structural features and sequence motifs of proline utilization A (PutA). Front Biosci (Landmark Ed) 17:556-68

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