Biosynthesis of Salvinorin A: Overexpression and Biochemical Characterization of Carboxy Methyltransferase from EST of Salvia divinorum Glands
LM Kutrzeba, JK Zjawiony, HJ Koo, E McDowell, A Laurentzi, FE Dayan
Planta Medica March 1, 2009 DOI: 10.1055/s-2009-1216489 (opens in new tab)
Study at a glance
AI-extracted from the abstract| Characteristics | Biochemical study Peer reviewed |
|---|---|
| Topics | Salvia divinorum |
| Citations | 1 |
| Key points | The cloned carboxymethyltransferase (CMT) methylated the C-18 carboxylic group of divinatorin A, divinatorin C, and hardwickiic acid, but not salvinorin A or B acids. The authors state this suggests CMT is substrate-specific and acts early in the salvinorin A biosynthetic pathway. |
Abstract
Abuse of unregulated substances by young adults has been a great concern of the US and international community. The active component of Salvia divinorum , salvinorin A ( 1 ) has a potent affinity to kappa opioid receptor in CNS. We studied the biosynthesis of this diterpenoid through the isolation of RNA and construction of cDNA library. Sequencing of the genetic material resulted in building an EST library containing all genes involved in biosynthetic assembly of 1 . We then cloned and overexpressed carboxy methyltransferase (CMT) gene in Escherichia coli to determine the substrate for the enzyme, and biochemically characterize it. We have employed 14 C-SAM, and five different substrates to test for the CMT activity in the cell free assay. We observed methylation of C-18 carboxylic group in divinatorin A, divinatorin C and hardwickiic acid, but not in highly oxygenated substrates like salvinorin A and B acids. This strongly suggests that CMT is substrate specific and that it is involved in the early stage of the pathway. Methyl esters of those substrates were independently synthesized to determine the products of the enzymatic reaction. Future work will involve purification of the enzyme and determination of K M and K CAT .