Metabolic enzymes in cells rarely function in isolation. Often their activities are coordinated by physical association with each other and cellular structures. A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu but rather are channeled between enzymes. Despite the widespread recognition that protein-protein interactions are ubiquitous, the mechanisms of substrate channeling remain relatively understudied and thus poorly understood. We help close this knowledge gap by exploring substrate channeling within and between the enzymes of proline catabolism. Proline catabolism comprises two enzymes and an intervening hydrolysis step. The flavoenzyme proline dehydrogenase (PRODH) catalyzes the oxidization of L-proline to ?1-pyrroline-5-carboxylate (P5C). Hydrolysis of P5C yields L-glutamate-?-semialdehyde, which is oxidized to L-glutamate by the NAD+-dependent enzyme P5C dehydrogenase (P5CDH). These enzymes have been implicated in many aspects of human health and disease, including tumor suppression, hyperprolinemia metabolic disorders, schizophrenia susceptibility, life- span extension, and the virulence of fungal and bacterial pathogens. In some organisms, PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA). The packaging of sequential enzymes from a metabolic pathway into a single protein not only implies substrate channeling but also the possibility of protein-protein interactions between the monofunctional enzymes. Thus, proline catabolism affords an excellent opportunity to compare substrate channeling within and between enzymes. The next phase of this project builds upon three major accomplishments made during the previous funding cycle: determination of the first crystal structures of PutA proteins, discovery of a novel hysteretic substrate channeling kinetic mechanism, and uncovering the first evidence for inter-enzyme substrate channeling between monofunctional PRODH and P5CDH enzymes.
The specific aims are to (1) elucidate the diverse structural solutions to substrate channeling that have evolved in the PutA family, (2) determine the structural basis and conservation of the hysteretic channeling mechanism, and (3) study substrate channeling in biological context by examining PRODH - P5CDH interactions in whole cells and determining the phenotypic consequences of disrupting proline metabolic channeling.
This project explores the molecular mechanism of communication between the two enzymes that catabolize the amino acid proline. Defects in proline catabolism cause hyperprolinemia disorders, and proline catabolism is important for tumor suppression in humans and contributes to the virulence of pathogens that have evolved to colonize proline-rich biological niches. Understanding how the activities of proline catabolic enzymes are coordinated enriches our understanding of the multifaceted roles of proline metabolism in human health and disease.
| Korasick, David A; White, Tommi A; Chakravarthy, Srinivas et al. (2018) NAD+ promotes assembly of the active tetramer of aldehyde dehydrogenase 7A1. FEBS Lett 592:3229-3238 |
| Korasick, David A; Campbell, Ashley C; Christgen, Shelbi L et al. (2018) Redox Modulation of Oligomeric State in Proline Utilization A. Biophys J 114:2833-2843 |
| Korasick, David A; Kon?itíková, Radka; Kope?ná, Martina et al. (2018) Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants. J Mol Biol : |
| 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; Tanner, John J (2018) Determination of protein oligomeric structure from small-angle X-ray scattering. Protein Sci 27:814-824 |
| 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 |
| Korasick, David A; Singh, Harkewal; Pemberton, Travis A et al. (2017) Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure. FEBS J 284:3029-3049 |
| 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 |
| Christensen, Emily M; Patel, Sagar M; Korasick, David A et al. (2017) Resolving the cofactor-binding site in the proline biosynthetic enzyme human pyrroline-5-carboxylate reductase 1. J Biol Chem 292:7233-7243 |
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