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Amorpha-4,11-diene synthase: Mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate

2005; Elsevier BV; Volume: 448; Issue: 1-2 Linguagem: Inglês

10.1016/j.abb.2005.07.015

1096-0384

S. Picaud, P. Mercke, Xiaofei He, Olov Sterner, Maria Brodelius, David E. Cane, Peter E. Brodelius,

Microbial Natural Products and Biosynthesis

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-2H]FPP, (1S)-[1-2H]FPP, and [1,1-2H2]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 differently 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 afford the final product (1S,6R,7R,10R)-amorpha-4,11-diene.