Elucidation of Reaction Mechanisms of Chain-elongation Enzymes in Isoprenoid Biosynthesis

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1. Site-directed mutations were introduced into thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus, and structure-reactivity relation was investigated. As a result, it has been demonstrated that Phe-220 and Gln-221 in the conserved motif FQXXDD are both essential for catalysis and that Lys-47 and Lys-183 are involved in the binding of isopentenyl diphosphate. Coupled together, these findings have led to a better understanding of the mechanism of prenyltransferase reaction.2. The genes for the two components of B.stearothermophilus heptaprenyl diphosphate synthase were cloned, sequenced, and overexpressed. The gene encoding a methyltransferease responsible for the final step in menaquinone biosynthesis was found to be located between the two genes for heptaprenyl diphosphate synthase.3. The purification of geranylgeranyl diphosphate synthase from bovine brain was achived, and the catalytic function was clarified.4. A novel type of protein modification by isoprenoid derived materials was discovered by investigating the metabolism of mevalonate in halobacterium halobium.5. Farnesol phosphokinase was found in Botryococcus braunii and its properties was disclosed.6. A new metabolic system for the conversion of dolichols and dehydrodolichols into their aldehydes was discovered.