A functional question in cell biology is how metabolic pathways are integrated and controlled to produce a balanced, efficient metabolism. The long term goal of our research is to gain a molecular understanding of regulatory and metabolic interactions amongst pathways. A solid understanding of such metabolic integration is essential to predict the response of cells to environmental change, develop metabolic modeling systems, and target metabolism for rational drug design and/or production of small molecules. As a model system to address these questions, we use the biosynthesis of thiamine in Salmonella typhimurium with a particular focus on the newly discovered Alternative Pyrimidine Biosynthetic (APB) pathway which synthesizes the pyrimidine moiety (HMP) of thiamine pyrophosphate (TPP). Objectives. In this proposal we will further characterize the biochemical steps in the APB pathway by: I) identifying metabolic precursors to the pyrimidine moiety of thiamine via this pathway, ii) determining the biochemical role of the ApbA and ApbC gene products involved in the APB pathway, and iii) investigating protein-protein interactions between Purl and a component of the APB pathway. To accomplish these objectives, modern chemical, biochemical, molecular biological and genetic techniques will be employed. Product. This comprehensive study of the biochemical and genetic components of the APB pathway will allow us to gain insights into a number of areas related to pathway integration. Molecular characterization of the APB pathway will further our understanding of the biosynthesis of an essential vitamin and define a central component of a model system for probing interactions among central metabolic pathways in the cell. Ultimately this work will contribute to our understanding of the global metabolic strategies that S. typhimurium uses to survive in its natural environment.
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