Thiamine pyrophosphate (vitamin B1) is the cofactor involved in the catalysis of bond cleavage and bond formation directly adjacent to carbonyl groups. In contrast to the wealth of information available on the mechanistic enzymology of thiamine dependent enzymes (e. g., pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, transketolase), the mechanistic enzymology of thiamine biosynthesis is still in its infancy. We propose to over-express and carry out mechanistic studies on several of the enzymes involved on this pathway. Our initial goal is to elucidate the mechanism of thiamine phosphate pyrophosphorylase, the enzyme that catalyzes the coupling of the thiazole and the pyrimidine moieties. Our longer term goal is to determine the mechanism of the thiazole and the pyrimidine assembly. Our strategy for studying the mechanism of thiamine phosphate pyrophosphorylase will initially involve the cloning, sequencing and over-expression of the thi B gene in E. coli. We propose three mechanisms for the coupling reaction; an SN2 mechanism, an SN1 mechanism and an addition-elimination mechanism. Experiments designed to differentiate between these mechanisms will include kinetic studies to determine if a ternary complex is formed, stereochemical studies to determine if the reaction proceeds with inversion or retention, isotope effect studies to test for an enzyme pyrimidine adduct and determination of the active site sequence using mechanism based inactivation. Thiamine is an essential vitamin in the human diet (RDA = 1.4mg/day). Its deficiency leads to a neurological disorder called beriberi. Thiamine is therefore included as an additive in many foods. The annual production of thiamine is 2,000 tons, of which is produced by total synthesis. Cloning of the thiamine biosynthetic genes therefore has potential applications in the commercial production of thiamine fermentation.
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