Synthetic, mechanistic, and structural studies on the enzymes of diterpenoid biosynthesis

Diterpenoids are derived from the universal precursor geranylgeranyl diphosphate, and exhibit a wide variety of biological functions. Taxol, a diterpenoid isolated first from the Pacific yew is one of the most potent anticancer drugs found today. Casbene, another diterpene hydrocarbon isolated from castor bean, shows antibacterial and antifungal activity. A better understanding of the biosynthetic steps of diterpenes is important for future engineering because of the extremely low yields in chemical syntheses and very low availability of these compounds from the plants.; This dissertation has focused on the biosynthetic enzymes from isopentenyl diphosphate to diterpenes, especially on taxadiene synthase, in order to set up convenient biosynthetic systems for formation of taxadiene, the key intermediate of taxol.; With 4-13C-isopentenyl diphosphate as starting substrate, enzymatic syntheses of GGDP, casbene and GGDP analogs have been explored by monitoring the carbon-flow by 13C NMR, leading to the production of GGDP and casbene in high yield.; Casbene synthase has been overexpressed as a highly soluble and active protein by removing the signal peptide predicted from homology and secondary structural studies after the incidental finding of deletions present in full-length protein. The signal peptide of taxadiene synthase has been similarly predicted and truncated, providing highly soluble and active protein, which solved the major obstacle for cell-free biosynthesis of taxadiene.; Taxadiene synthase was shown to catalyze the formation of taxa-4(5), 11(12)-diene from GGDP, together with 13% of taxa-4(20), 11(12)-diene. Substrate specificity studies have been done on truncated taxadiene synthase, and several analogs were demonstrated to be artificial substrates. Interestingly, truncated taxadiene synthase was proved to be a sesquiterpene cyclase, yielding four products from farnesyl diphosphate.; Extensive N-terminal truncations and mutations have been performed on taxadiene synthase, and two residues Y89 and H90 were shown to be crucial for activity. Site-directed mutagenesis has been performed on several "odd" residues at the terminal of taxadiene synthase, and interesting results have been obtained.; Both in vitro chemoenzymatic synthesis and in vivo production of taxadiene have been successfully carried out. Taxadiene was subjected to chemical oxidation by selenium dioxide and singlet oxygen.