Cyclic terpenoids are found throughout nature but comprise an especially important class of compounds from plants which serve as pharmaceutical agents with antibiotic, antifungal and antitumor activities. The biosynthesis of cyclic terpenes is determined by key branch point enzymes referred to as terpene cyclases, or more properly, terpene synthases. The objective of this proposal is to understand as completely as possible the structural, functional, and chemical features governing the cyclizations of farnesyl diphosphage (FPP) catalyzed by highly homologous plant sesquiterpene cyclases and one diterpene cyclase. The proposed work relies on our recent success with a domain-swapping strategy to map and substitute functional domains between sesquiterpene cyclase genes, and enzymological characterization of the bacterial expressed cyclase proteins. We are now proposing to use new crystallographic data of the wildtype and mutant enzymes in an iterative process with domain swapping and site directed mutagenesis to identify, in much greater detail, those domains and amino acid residues essential for catalysis of particular partial steps within the cyclase reactions. The proposed experimental plan will provide the foundation for our long range goal that focuses on a rationally based redesign of terpene cyclases for the enzymatically directed synthesis of pharmaceutically important terpenoids or their synthetic precursors. This structure/function analysis should also increase our fundamental appreciation for terpene enzymology and biosynthesis in general, and the biosynthesis of cyclic terpenoids like cholesterol, steroid hormones, bile acids, carotenoids and retinoids, and lipid soluble vitamins, A,D,E, and K in particular.
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