This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project is a multi-faceted study on the regulation of the proline utilization A (PutA) flavoprotein from Escherichia coli. PutA combines DNA-binding and proline catabolic activities into a single polypeptide (1320 residues) to regulate proline utilization in E. coli. In the cytoplasm, PutA represses transcription of the proline utilization (put) regulon that encodes the putP (a proline transporter) and putA genes by binding to the put control DNA region. To perform its catalytic functions, PutA peripherally associates with the membrane where it oxidizes proline to glutamate in two successive steps using separate proline dehydrogenase and ?1-pyrroline-5-carboxylate dehydrogenase domains. Our overall goal is to determine the mechanism by which PutA changes its intracellular location and switches from a DNA-binding transcriptional repressor to a membrane bound enzyme. Recently we uncovered a novel redox mechanism for regulating protein function and gene expression. We have demonstrated that PutA-membrane interactions are highly dependent on the flavin redox state. Using surface plasmon resonance we showed that flavin reduction induces tight PutA-membrane binding (Kd 0.01 nM, pH 7.4) in stark contrast to oxidized PutA that lacks membrane binding activity. We have built a thermodynamic model for the regulation of PutA in which the expression of put genes is activated by reduction of the flavin and subsequent binding of PutA to the membrane. We are also developing new projects on PutA from gastrointestinal bacterial pathogens such as Helicobacter and on proline dehydrogenase (PRODH) from eukaryotes with the goal of examining the role proline has in balancing the overall intracellular redox environment in various organisms. Some of this work is in collaboration with Dr. Martin Dickman in the Department of Plant Pathology at the University of Nebraska-Lincoln. So far, we have shown that in yeast and mammalian cultured cells overexpression of PRODH and subsequent lowering of intracellular proline levels increases sensitivity of cells to oxidative st
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