BIOL 117 |
| The biosynthesis of thiamin-thiazole in eukaryotes is different from the prokaryotic biosynthesis and is poorly understood. Compared to five thiazole biosynthetic proteins in prokaryotes, only one gene has been identified in eukaryotes (THI4 in Saccharomyces cerevisiae) to be responsible for thiazole biosynthesis. THI4 was overexpressed in E. coli and purified. We identified three adenylated metabolites tightly bound to purified THI4. Characterization of the major species revealed it to be an adenylated thiazole derivative. The unexpected structure of this molecule, yielded the first critical insights into the mechanism of thiazole biosynthesis in eukaryotes and demonstrated that NAD is the substrate for THI4. The crystal-structure of the enzyme shows the characterized product bound to the active site. Extensive mutagenesis was done to identify a set of mutants, which were partially active and allowed the identification of the substrate (NAD) and the characterization of the initial steps of thiazole biosynthesis. We have also identified an enzyme THI6, annotated as a thiazole kinase/thiamin phosphate synthase that can convert the product of THI4 to thiamin phosphate in the presence of hydroxymethylpyrimidine, pyrophosphate and Mg+2.
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Poster Session
8:00 PM-10:00 PM, Monday, August 20, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |