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

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Author

  • S Picaud
  • P Mercke
  • XF He
  • Olov Sterner
  • M Brodelius
  • DE Cane
  • P E Brodelius

Summary, in English

Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-H-2]FPP, (1S)-[1-H-2]FPP, and [1,1-H-2(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 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. (c) 2005 Elsevier Inc. All rights reserved.

Publishing year

2006

Language

English

Pages

150-155

Publication/Series

Archives of Biochemistry and Biophysics

Volume

448

Issue

1-2

Document type

Journal article

Publisher

Academic Press

Topic

  • Organic Chemistry

Keywords

  • GC-MS
  • proton NMR
  • COSY
  • deuterated substrate
  • enzyme mechanism
  • sesquiterpenes
  • 11-diene synthase
  • amorpha-4

Status

Published

ISBN/ISSN/Other

  • ISSN: 0003-9861