P-57 完全^<13>C標識プレニル二リン酸の酵素的合成と二次元^<13>C-NMR解析(ポスター発表の部)

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Terpenoids are widely distributed in nature, including hormones that regulate life cycles of mammals, insects and plants. Preparation of useful labeled-compounds makes powerful tools for studying comprehensive analysis and biosynthesis of terpenoids. Here, we synthesized fully ^<13>C-labeled prenyl diphosphates using enzyme cocktails which contain recombinant enzymes responsible for biosynthesis of farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP) from mevalonate. Six cDNAs encoding mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate decarboxylase (DPMVD), isopentenyl diphosphate isomerase (IPI), FDP synthase (FPS) and GGDP synthase (GGPS) were cloned from Neurospora crassa and expressed in E. coli to produce recombinant enzymes as His-tagged proteins. These enzymes were purified by Ni-NTA affinity chromatography and combined with a reaction buffer containing cofactors and the substrate [U-^<13>C_6]mevalonate (97% ^<13>C-label). The ^<13>C-labeled mevalonate was prepared by secretion into the fermentation broth of Saccharomycopsis fibuligera, which was incubated with a [U-^<13>C_6]glucosecontaining medium. The enzyme cocktail containing five enzymes (MVK, PMVK, DPMVD, IPI and FPS) produced ^<13>C-labeled FDP from^<13>C-labeled mevalonate without dilution by natural abundance. Furthermore, uniformly ^<13>C-labeled GGDP was successful ly synthesized in the presence of GGPS into the above enzyme cocktai The ^<13>C-labeled FDP was analyzed by GC-MS, ion-pair HPLC-ESI-MS and two-dimensional ^<13>C-NMR (C-C COSY). The NMR signals were obtained from only 1.3mg of the sample and easily assigned on the basis of the cross-peaks by C-C COSY. We are synthesizing ^<13>C-labeled mevalonate and prenyl diphosphates from 99.9% ^<13>C-labeled acetate in vitro.
天然有機化合物討論会の論文
2007-08-24

P-57 完全^<13>C標識プレニル二リン酸の酵素的合成と二次元^<13>C-NMR解析(ポスター発表の部)

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