Dissertation
(–)-KOLAVENYL DIPHOSPHATE SYNTHASE AND ACETYLTRANSFERASE INVOLVED IN SALVINORIN A BIOSYNTHESIS PATHWAY IN SALVIA DIVINORUM
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/117504
Abstract
Salvia divinorum (Lamiaceae), a plant native to Oaxaca in Mexico, has been traditionally used by Mazatecs in spiritual practices due to its hallucinogenic property. A clerodane diterpenoid salvinorin A is found to be the principle psychoactive constituent in S. divinorum and identified as a selective and potent κ opioid receptor agonist. The unique structure and potential mechanism as opioid receptor ligand make salvinorin A a promising template to develop analogues as novel agents to treat pain, drug abuse and other mental disorders with reduced side effects. Elucidation of the virtually unknown biosynthetic pathway of salvinorin A will facilitate its production and structural diversification for research and medical applications.
The main goals of my dissertation are: 1) to identify and characterize the enzyme catalyzing the first committed reaction, the formation of (-)-kolavenyl diphosphate ((-)-KPP), which is further dephosphorylated into kolavenol as the first isolated putative intermediate in the salvinorin A biosynthesis pathway. 2) to investigate the nature of the enzymatic reaction responsible for acetylating the C-1 and C-2 hydroxyl groups of salvinorin H, salvinorin D and salvinorin E in S. divinorum.
Trichomes were identified as the major site of salvinorin accumulation in S. divinorum. Functional characterization of a class II diTPS expressed in trichome designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation studies of SdKPS confirmed critical structural elements that determine the product specificity of this enzyme.. Formation of salvinorin D and salvinorin E from salvinorin H and salvinorin C from salvinorin D or salvinorin E were detected in enzyme activity assays performed on crude trichome protein extracts using acetyl-CoA with salvinorins as cosubstrates. Acetyltransferase activities toward salvinorins were highest in trichome. Candidate genes of these acetyltransferases were predicted to be BAHD acyltransferase members and isolated from trichome specific transcriptome for protein expression and functional characterization. Our study improved our understanding of salvinorin A biosynthesis and provided more information needed for future studies of identifying biosynthetic enzymes involved in this pathway.
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Details
- Title
- (–)-KOLAVENYL DIPHOSPHATE SYNTHASE AND ACETYLTRANSFERASE INVOLVED IN SALVINORIN A BIOSYNTHESIS PATHWAY IN SALVIA DIVINORUM
- Creators
- Xiaoyue Chen
- Contributors
- David R Gang (Advisor)Sanja Roje (Committee Member)Bernd M Lange (Committee Member)Eric H Roalson (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 125
- Identifiers
- 99900581709701842
- Language
- English
- Resource Type
- Dissertation