Archives of Biochemistry and Biophysics 448 (2006) 150–155 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2005.07.015 Amorpha-4,11-diene synthase: Mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate Sarah Picaud a , Per Mercke a , Xiaofei He b , Olov Sterner c , Maria Brodelius a , David E. Cane b , Peter E. Brodelius a,¤ a Department of Chemistry and Biomedical Science, University of Kalmar, SE-39182 Kalmar, Sweden b Department of Chemistry, Brown University, Providence, Rhode Island 02912-9108, USA c Department of Organic Chemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden Received 4 June 2005, and in revised form 15 July 2005 Available online 11 August 2005 Abstract Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deute- rium-labeled farnesyl diphosphates, (1R)-[1- 2 H]FPP, (1S)-[1- 2 H]FPP, and [1,1- 2 H 2 ]FPP. GC–MS analysis of amorpha-4,11-diene formed from the deuterated FPPs shows that the deuterium atoms are retained in the product. Furthermore, analysis of the MS- spectra obtained with the diVerently labeled substrate indicates that the H-1si-proton of FPP is transferred during the cyclization reaction to carbon 10 of amorphadiene while the H-1re-proton of FPP is retained on C-6 of the product. Proton NMR and COSY experiments proved that the original H-1si-proton of FPP is located at C-10 of amorpha-4,11-diene as a result of a 1,3-hydride shift following initial 1,6-ring closure. The results obtained support the previously suggested mechanism for the cyclization of farnesyl diphosphate by amorph-4,11-diene synthase involving isomerization of FPP to (R)-nerolidyl diphosphate (NPP), ionization of NPP, and C-1,C-6-ring closure to generate a bisabolyl cation, followed by a 1,3-hydride shift, 1,10-ring closure to generate the amorphane skeleton, and deprotonation at either C-12 or C-13 to aVord the Wnal product (1S,6R,7R,10R)-amorpha-4,11-diene.  2005 Elsevier Inc. All rights reserved. Keywords: Sesquiterpenes; Amorpha-4,11-diene synthase; Enzyme mechanism; Deuterated substrate; GC–MS; Proton NMR; COSY Sesquiterpenoids are a structurally diverse class of iso- prenoids found in plants, fungi, and some bacteria, which play a variety of physiological and ecological roles, includ- ing plant–plant, plant–insect, and plant–pathogen interac- tions. The structural diversity and stereochemical complexity of the C 15 -isoprenoid skeletons of sesquiterpe- noids are remarkable. More than 300 types of cyclic ses- quiterpenes have been characterized to date and each is derived from the common acyclic precursor, farnesyl diphosphate (1, FPP). 1 Sesquiterpene synthases are key branch point enzymes in the biosynthesis of these com- pounds. Several sesquiterpene synthases have been cloned from plants and the deduced amino acid sequences show high identity/similarity to one another [1–9]. Numerous plant sesquiterpene synthase sequences have been recog- nized on the basis of this sequence similarity, but the nature of the cyclization products is as yet unknown. Our understanding of the relationship between protein struc- ture and product selectivity of highly homologous sesqui- terpene synthases is still limited. We recently reported the molecular cloning and bio- chemical characterization of amorpha-4,11-diene synthase from Artemisia annua, which catalyzes the cyclization of farnesyl diphosphate to (1S,6R,7R,10R)-amorpha-4,11- * Corresponding author. Fax: +46 480 446262. E-mail address: peter.brodelius@hik.se (P.E. Brodelius). 1 Abbreviations used: ADS, amorpha-4,11-diene synthase; AMU, atomic mass unit; FPP, farnesyl diphosphate; GPP, geranyl diphos- phate; NPP, nerolidyl diphosphate; IMAC, immobilized metal aYnity chromatography.