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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.