The mechanistic enzymology of thiamin biosynthesis

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DESCRIPTION (provided by applicant): Thiamine is an essential component of the human diet and is added to many commercial foods (RDA = 1.4 mg). Deficiency in this vitamin results in the neurological diseases beriberi and Wernicke encephalopathy. At the molecular level, thiamine dependent enzymes play a particularly important role in carbohydrate metabolism and include transketolases, alpha-ketoacid decarboxylases, alpha-ketoacid oxidases and acetolactate synthase. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamine biosynthesis in both prokaryotes and eukaryotes. Our studies are significant for three reasons. First, since this vitamin is a required component of the human diet and an essential cofactor for all forms of life, it is important to understand how it is biosynthesized. Second, from the perspective of basic science, the biosynthetic pathway involves an unusually large amount of unprecedented chemistry: The mechanism of the complex oxidative condensation reaction to give the thiazole and the remarkable rearrangement involved in the conversion of AIR to the pyrimidine have not yet been elucidated. Finally, our studies will facilitate the construction of overexpression strains that will be of use for the commercial production of thiamine or its components (3,500 tons/year produced by total synthesis). In this grant period, we will carry out mechanistic studies on the thiazole and the pyrimidine forming reactions, initiate studies on thiamine biosynthesis in yeast, screen a peptide library for inhibitors of thiamine biosynthesis and evaluate the use of thiamine as a selective proteome probe.