Amorpha-4,11-diene synthase: mechanism and stereochemistry of the enzymatic cyclization of farnesyl diphosphate.
Recombinant amorpha-4,11-diene synthase from Artemisia annua, expressed in Escherichia coli, was incubated with the deuterium-labeled farnesyl diphosphates, (1R)-[1-(2)H]FPP, (1S)-[1-(2)H]FPP, and [1,1-(2)H2]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.
Picaud
Sarah
S
Department of Chemistry and Biomedical Science, University of Kalmar, SE-39182 Kalmar, Sweden.
Mercke
Per
P
He
Xiaofei
X
Sterner
Olov
O
Brodelius
Maria
M
Cane
David E
DE
Brodelius
Peter E
PE
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031172:Artemisia annua / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012717:Sesquiterpenes; D013237:Stereoisomerism
D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase