Identification of the diterpene synthase in Salvia divinorum used to synthesize salvinorin A

Adrian LaurenziTucson High Magnet SchoolResearch MethodsMarch 26, 2008

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.

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)

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)

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

Salvinorin A quantification

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

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

BLAST searching & assemblyBLAST 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

BLAST searching & assembly

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

Acknowledgments

Margaret Wilch

Kevin Vogel

Dr. David Gang, Ph.DDepartments of Plant Sciences,Biochemistry, and Molecular BiophysicsBIO5 InstituteUniversity of Arizona