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Exploration of Redox-Based Functional Switching and Intermediate Substrate Channeling in Proline Catabolism
The bioaccumulation and catabolism of the amino acid proline have been shown to participate in numerous cellular processes. There is a wealth of emerging evidence that proline metabolism plays vital roles in a number of different pathogenic organisms; ranging from energy production to stress protection and beyond, as reviewed in Chapter 1. In eukaryotes and gram-positive bacteria, the catabolism of proline to form glutamate is catalyzed by two separate enzymes, proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). In gram-negative bacteria, these two enzymes are fused into a single, multifunctional protein known as proline utilization A, or PutA. The formation of glutamate from proline is enzymatically intriguing due to the phenomena of redox-based functional switching and substrate channeling. Some PutA proteins exhibit a third functionality in addition to the typical PRODH and P5CDH activities. These trifunctional PutAs oscillate between functioning as a self-regulating transcriptional repressor and a membrane-bound enzyme, depending on the redox state of the flavin cofactor of the PRODH active site. Chapter 2 examines the role of the c-terminus of Escherichia coli PutA (EcPutA) in membrane binding, and establishes the region of PutA-membrane interactions. Chapter 3 furthers the knowledge of the mechanism of functional switching, highlighting key residues vital for the transmission of the redox-based signal from the flavin cofactor to the membrane binding domain and discusses the development of assays to examine signal transmission in vivo. Substrate channeling occurs when an intermediate substrate is transferred between two enzymes or two active sites, without equilibrating into the surrounding environment. Previous evidence demonstrates that this phenomenon occurs via a hollow cavity in PutA proteins and also occurs between monofunctional PRODH and P5CDH in vitro. Chapter 4 of this dissertation examines P5C channeling in mono- and multifunctional systems in vivo. Furthermore, substrate channeling was disrupted both between monofunctional PRODH and P5CDH from Thermus thermophilus and in PutA proteins through site-directed mutagenesis. Collectively, the research presented in this dissertation has made significant advancements in the knowledge of functional switching and substrate channeling in monofunctional and multifunctional enzymes of proline metabolism.
Christgen, Shelbi Lynn, "Exploration of Redox-Based Functional Switching and Intermediate Substrate Channeling in Proline Catabolism" (2017). ETD collection for University of Nebraska-Lincoln. AAI10616683.