74. 酵素合成法による完全C-13標識化と多次元NMRを組合せたmiltiradiene合成酵素の機能解析(口頭発表,植物化学調節学会第46回大会)

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Diterpenes show various chemical structures and diverse physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) catalyze successive two-step type-B and type-A reaction of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes, with the majority being uncharacterized. The cDNA from S. moelledorffii encoding BDTC-like enzyme (SmMDS) was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with the molecular mass of 272. Mutation experiments showed that the intermediate of the reactions was (+)-CDP. When the diterpene hydrocarbon was enzymatically synthesized from [U-^<13>C_6]mevalonate, all carbons were labeled with ^<13>C-stable isotope (>99%). The fully ^<13>C-labeled product was subjected to ^<13>C-^<13>C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multi-dimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a biosynthetic precursor of tanshinones identified from the Chinese medicinal herb, Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii.
2011-10-03