Post on 29-Jan-2016
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