P-89 完全C-13標識された多環性テルペノイドの酵素的合成(ポスター発表の部)

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Terpenoids are widely distributed in nature. They include hormones that regulate life cycles of mammals, insects and plants. Preparation of labeled-compounds will be powerful tools for studying comprehensive analysis and biosynthesis of terpenoids. We synthesized fully C-13 labeled mevalonate with enzyme cocktails which contain recombinant four enzymes responsible for mevalonate biosynthesis from acetate, cofactors, and the substrate [U-^<13>C_2) acetate (99% C-13 label). The mass spectra of C-13 labeled mevalonolactone, as dehydration form of mevalonate, showed no dilution with natural abundance peaks. The NMR signals showing C-C coupling were observed from 0.7mg of the sample. To produce polycyclic terpenoids from acetate in one test tube, these four enzymes were combined with an enzyme cocktail for synthesis of prenyl diphosphate from mevalonate, and terpene cyclase. For example, amorphadiene is a biosynthetic intermediate of antimalarial reagent artemisinine. The amorphadiene is biosynthesized from farnesyl diphosphates by amorphadiene synthase, a sesquiterpene cyclase in Artemisia annua L. We cloned amorphadiene synthase open reading frame and produced its recombinant protein in E. coli. This cyclase protein was added into the enzyme cocktail responsible for labeled farnesyl diphosphates synthesis, and uniformly C-13 labeled amorphadiene was synthesized in one test tube. Similarly, addition of bifunctional ent-kaurene synthase protein into the enzyme cocktail synthesizing geranylgeranyl diphosphates led [U-^<13>C_<20>] ent-kaurene production. Thus, we established the basis of enzymatic synthesis of fully C-13 labeled multicyclic terpenoids in one test tube. These labeled probes will be used as functional analysis of unknown genes and comprehensive analysis of terpenoid biosynthetic pathways.
天然有機化合物討論会の論文
2008-09-01

P-89 完全C-13標識された多環性テルペノイドの酵素的合成(ポスター発表の部)

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