Abstract
The complete genome of a lycophyte, Selaginella moellendorffii, has been sequenced and been available for extended research of functional genomics. S. moellendorffii genome contains putative 13 diterpene cyclase genes that are homologus to the angiosperm monofunctional cyclases and the moss and gymnosperm bifunctional cyclases. We cloned a possible diterpene cyclase gene SmBDTC that encodes a bifunctional enzyme catalyzing cyclization reaction of geranylgeranyl diphosphate (GGDP) to hydrocarbon product(s). The recombinant protein of SmBDTC expressed in Escherichia coli produced a single hydrocarbon product with the molecular mass of 272 from GGDP. A mutant of SmBDTC lacking ionization-initiated cyclization activity afforded copalyl diphosphate from GGDP. Although another mutant lacking protonation-initiated cyclization activity recognized both syn- and (+)-copalyl diphosphates, (+)-copalyl diphosphate as the native substrate of SmBDTC was determined. The SmBDTC product was enzymatically synthesized from [U-^<13>C_6] mevalonic acid, and all carbon atoms were fully labeled with ^<13>C isotope (>99%). The inverse gated-decoupling ^<13>C NMR spectrum of fully ^<13>C labeled compound confirmed 20 garbon signals. Two dimensional NMR spectrum (C-C COSY) and three-dimensional HCCH COSY spectrum demonstrated that SmBDTC catalyzed the production of miltiradiene, a biosynthetic precursor of tanshinones identified from the Chinese medicinal herb Salvta miltiorrhiza.
