28. 標識イソプレノイド化合物の酵素的合成

概要

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All terpenoids are derived from dimethylallyl diphosphate (DMAPP) and/or isopentenyl diphosphate (IPP) in organisms. Isotopelabeled DMAPP and IPP will be powerful tools for screening and biosynthetic studies of terpenoid compounds. Here, we report the enzymatic preparation of the isoprenoid compound, farnesyl diphosphate (FDP). [U-^<13>C_6] mevalonic acid (MVA) was prepared by incubation of [U-^<13>C_6] glucose with an yeast Saccharomycopsis fibuligera. To synthesize FDP from MVA, five enzymes are involved; mevalonate kinase (MVK), phosphomevalonate kinase (PMVK), diphosphomevalonate decarboxylase (DPMVDC), IPP isomerase (IPPI) and farnesyl diphosphate synthase (FPS). cDNAs encoding these enzymes were cloned depending on the information of genome database of Neurospora crassa. Each open reading frame of these cDNAs was ligated into pQE30 vector to produce the recombinant proteins in E. coli. Recombinant proteins of MVK, DPMVDC, IPPI and FPS were produced as soluble proteins and recovered from E. coli cells. Since recombinant PMVK was produced as the inclusion body, the ORF was ligated into pGEX vector to produce it as the glutathion-S-transferase fusion protein. Enzymatic preparation of FDP, DMAPP and IPP are in progress.
植物化学調節学会の論文
2005-10-13