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Planta Med 2009; 75 - P-51
DOI: 10.1055/s-2009-1216489

Biosynthesis of Salvinorin A: Overexpression and Biochemical Characterization of Carboxy Methyltransferase from EST of Salvia divinorum Glands

LM Kutrzeba 1, JK Zjawiony 1, 2, HJ Koo 3, E McDowell 3, A Laurentzi 3, DR Gang 3, FE Dayan 4
  • 1Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA
  • 2National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA
  • 3University of Arizona, Department of Plant Sciences and BIO5 Institute, Tucson, AZ 85721, USA
  • 4Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University, MS 38677, USA

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 14C-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 KM and KCAT.