Biochemistry, Biophysics and Molecular Biology, Roy J. Carver Department of
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It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral entkaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, we applied a synthetic biology approach to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a “splicing” error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, we can assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally novel enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.
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Jackson, Alana J.; Hershey, David M.; Chesnut, Taylor; Xu, Meimei; and Peters, Reuben J., "Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization" (2014). Biochemistry, Biophysics and Molecular Biology Publications. 88.