1. Identification of the diterpene synthase in Salvia divinorum used to synthesize salvinorin A Adrian Laurenzi Tucson High Magnet School Research Methods March 26, 2008

2. What is Salvia divinorum? Cultigen found in Sierra Mazatec region of Oaxaca, Mexico Traditionally used by the Mazatec Indians for its hallucinogenic and therapeutic effects.

3. Why are we interested in it? Produces salvinorin A; a potent k-opioid receptor agonist Medicinal potential: Diseases w/ hallucinations: schizophrenia, dementia, Alzheimer's, depression (Roth et al., 2002) Drug addiction (Roth et al., 2002) HIV and AIDS (Peterson et al., 2001)

4. Localization of salvinorin A Salvinorin A is a diterpenoid found in glandular trichomes Salvinorin A is likely produced within glandular trichomes Possible anti-feeding properties (insect or mammal herbivores)

5. Purpose Identify the protein that acts as the first step in the biosynthesis of salvinorin A Future applications: ● Synthesis of salvinorin A ● Further understanding of chemical mechanisms of k-opioid receptor in the brain

7. Salvinorin A quantification

8. Biosynthesis of salvinorin A IPP and DMAPP are synthesized via the non-mevalonate (MEP) pathway (Kutrzeba et al., 2007) GGPP is assembled by joining 4 units of IPP A diterpene synthase converts GGPP into a precursor of salvinorin A

9. cDNA library & sequencing The cDNA library was sequenced via 454 sequencing RNA was isolated from S. divinorum trichomes and used to construct a cDNA library

10. BLAST searching & assembly BLAST diterpene synthases against S. divinorum library Results assembled into a small contig, which is then Netblasted Contig1 assembled from 112 fragments and Netblasted BLAST results from Netblast against S. divinorum library

11. BLAST searching & assembly

12. Conclusion Enzyme likely to be an ent-copalyl diphosphate synthase Future methods: Express protein and assay with GGPP Test for enzyme activity in S. divinorum

13. Acknowledgments Margaret Wilch Kevin Vogel Dr. David Gang, Ph.D Departments of Plant Sciences, Biochemistry, and Molecular Biophysics BIO5 Institute University of Arizona