Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization.

摘要: It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, ent-trachylobane, addition ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity underlying reaction catalyzed by KSs, ent-beyerene ent-trachylobane synthases not yet been identified. Given our interest LRD biosynthesis, recent availability genome sequence, a synthetic biology approach applied biochemically characterize KS(-like) enzymes [KS(L)s] found Ricinus communis [i.e., RcKS(L)s]. In particular, bacteria engineered relevant ent-copalyl diphosphate precursor genes based on predicted RcKS(L)s, although this ultimately correction "splicing" error one genes, highlighting dependence such accurate sequences. Nevertheless, it is possible assign each RcKS(L)s observed activities, providing access functionally enzymes. Intriguingly, product distribution seems support distinct mechanism proposed quantum chemical calculations, rather than classically pathway.