Investigating reactions catalyzed by terpene synthases in a novel model system.

Terpene synthases are a set of enzymes that initiate the biosynthetic production of the largest class of known natural products. However, not much is known about how these enzymes carry out their complex cyclization reactions to form polycyclic olefins from linear hydrocarbon diphosphates such as geranylgeranyl diphosphate. Since rice is known to produce many diterpenoid natural products and the rice genome was recently published, sequence information for many putative diterpene synthases was available. This provided the basis for functional characterization of the diterpene synthases from rice (Oryza sativa ssp. Indica). An enzyme producing syn-pimaradiene was identified, followed by those producing syn-stemodene, ent-kaurene, ent-isokaurene, ent-sandaracopimaradiene, ent-cassadiene, and syn-stemarene. At the same time another group published similar results, however one of the reported ent-isokaurene synthases was reported as ent-pimaradiene synthase by another research group. Noting this difference in product with only the three amino acid differences in the active site, experiments were carried out leading to discovery of a single amino acid residue that switches product profile completely to pimaradienes from kaurenes, OsKSL5i:I664T. This switch was found not only in the originally targeted isokaurene synthase, OsKSL5, but also in the second reported isokaurene synthase from rice and kaurene synthases from rice and Arabidopsis. Further expounding on this idea, a similar amino acid difference was noted in diterpene synthases of conifers. This amino acid difference, A723S in abietadiene synthase, was a switch, too, with the product profile switching from >95% abietanes to >95% pimaranes. Utilizing a combination of functional genomics, metabolic engineering, macromolecular modeling, and enzyme biochemistry a basis for further investigations into how terpene synthases work has been provided.