Abstract
ent-Kaurene is a diterpene hydrocarbon compound and an intermediate of plant hormone gibberellins. This diterpene is biosynthesized from GGDP via ent-CDP. In flowering plants, two distinct cyclases, CPS and KS, are involved in the biosynthesis of ent-kaurene. Interestingly, lower vascular plants such as moss and liverwort biosynthesize ent-kaurene by bifunctional ent-kaurene synthases having both CPS and KS activities in single polypeptides. On the other hands, little is known about ent-kaurene synthetic genes and enzymes in ferns. Here, we cloned cDNAs of diterpene cyclases from Selaginella moellendorffii, and analyzed their function. Expression analysis showed that four genes were expressed during the growth of S. moellendorffii, which has eight diterpene cyclase genes in the genome. These recombinant proteins were produced by in vitro protein expression kit and used for functional analysis. SmDTC1 has DxDD motif and converted GGDP to ent-kaurene in the presence of Arabidopsis KS protein. SmDTC7 has DDxxD motif and converted GGDP to ent-kaurene in the presence of SmDTC1. These results indicated that SmDTC1 and SmDTC7 are CPS and KS, respectively. Interestingly, SmDTC3 used GGDP as substrate and slightly produce ent-kaurene. Hence, we concluded that SmDTC3 is a bifunctional ent-kaurene synthases, SmCPS/KS. From the mass spectrum analysis, SmDTC4 synthesized a novel diterpene hydrocarbon. The structure determination of the product by SmDTC4 is in progress.
