The mechanistic enzymology of thiamin biosynthesis

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Thiamin is an essential component of the human diet with an RDA of 1.2 mg. It is also an important commercial chemical and it is widely used as a food additive and flavoring agent. Annual production, by chemical synthesis, is on the order of 3,300 tons. Thiamin-dependent enzymes play an important role in carbohydrate and branched-chain amino acid metabolism. At this time, our mechanistic understanding of this class of enzymes is sophisticated. In contrast, while thiamin was the first vitamin identified, our understanding of its biosynthesis is still incomplete because the biosynthetic pathway is complex and involves unprecedented reaction chemistry. The long-term goal of our research is the complete mechanistic understanding of the enzymology of thiamin biosynthesis, salvage and catabolism, in all forms of life and the elucidation of connections between thiamin metabolism and other aspects of cellular physiology. In the next grant period, we describe studies designed to elucidate the biosynthesis of the thiamin thiazole and pyrimidine in both bacteria and eukaryotes. These heterocycle forming reactions involve novel biological chemistry. Our strategy will involve a combination of kinetics, modified substrates, protein mutagenesis and structural studies. We will also identify the genes involved in thiamin breakdown and salvage. Bacteria, in the human digestive tract, containing these genes are likely to play an important role in controlling the human thiamin RDA. Finally, we will explore the use of 18F-thiamin as a PET imaging agent, ultimately to determine the global thiamin distrubution in healthy and diseased humans. RELEVANCE (See instructions): Thiamin (vitamin B1) is essential for all forms of life. Our studies will facilitate the commercial production of thiamin by fermentation and have potential applications in the design of new antibiotics. Understanding thiamin catabolism and salvage will enable us to evaluate the importance of these activities in the human digestive tract. 18F-thiamin has potential use, as an imaging agent, in basic human biology and in tumor detection.