A fundamental biological question is how metabolic pathways are integrated and controlled to produce an efficient metabolism. The long-term goal is to contribute to the overall understanding of metabolic integration. Salmonella typhimurium is used for these studies because of its well-characterized genetic system, the advanced understanding of biosynthetic pathways in this organism and the availability of the annotated genome sequence of the close relative E. coli. A model system to study the interaction of metabolic pathways with the biosynthetic pathway of thiamine has been developed in this organism. In this proposal the metabolic needs for the synthesis of the hydroxymethyl pyrimidine (HMP) moiety will be clarified as will be the differing synthetic requirements dependent upon the level of flux through the purine biosynthetic pathway. This will be accomplished by: 1) determining the role of the ThiC protein in HMP formation, 2) identifying the metabolic role of the ApbC protein and explaining why it is required in thiamine synthesis, 3) determining the role of CoA in thiamine synthesis and 4) identifying the metabolic source of phosphoribosylamine in the absence of the PurF protein. These objectives will be accomplished with modern chemical, biochemical, and molecular biological and genetic techniques. The work will increase the understanding of the metabolic needs for HMP synthesis in different genetic backgrounds. In addition, it has the potential to uncover novel aspects of metabolism by probing gene product functions that are indirectly involved in thiamine synthesis. This type of work is critical as genome sequences are completed and ORF families are uncovered that cannot be functionally annotated by sequence alone. Ultimately this work will contribute to understanding of global metabolic strategies employed by bacteria to maintain an efficient metabolism.
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