Cyclization Mechanism of Amorpha-4,11-diene Synthase, a Key Enzyme in Artemisinin Biosynthesis

Abstract

Cyclization of farnesyl diphosphate into amorpha-4,11-diene by amorpha-4,11-diene synthase (ADS) initiates biosynthesis of artemisinin, a clinically important antimalarial drug precursor. Three possible ring-closure mechanisms, two involving a bisabolyl carbocation intermediate followed by either a 1,3-hydride shift or two successive 1,2-shifts, and one involving a germacrenyl carbocation, were proposed and tested by analyzing the fate of farnesyl diphosphate H-1 hydrogen atoms through (1)H and (2)H NMR spectroscopy. Migration of one deuterium atom of [1,1-(2)H(2)]farnesyl diphosphate to H-10 of amorpha-4,11-diene singled out the bisabolyl carbocation mechanism with a 1,3-hydride shift. Further confirmation was obtained through enzyme reactions with (1R)- and (1S)-[1-(2)H]farnesyl diphosphate. Results showed that deuterium of the 1R compound remained at H-6, whereas that of the 1S compound migrated to H-10 of amorpha-4,11-diene. Incorporation of one deuterium into amorphadiene in the cyclization process was observed when the reaction was performed in (2)H(2)O, as evidenced by an increase of 1 amu in the mass of the molecular ion.

Expression of a synthetic Artemesia annua amorphadiene synthase in Aspergillus nidulans yields altered product distribution

“…Thus it would be expected to Wnd germacrene and eudesmane as co-products of AriS; expression of AN3277.3 cDNA in Saccharomyces did indeed yield these sesquiterpenes along with aristolochene (Supplementary Data). Likewise AD and bergamotene are synthesized via the bisabolyl cation 17 [12,34,35], hence bisabolene and bergamotene are not unlikely co-products; the farnesenes are simply uncyclized FPP.…”

Increased Accumulation of Artemisinin and Anthocyanins in Artemisia annua Expressing the Arabidopsis Blue Light Receptor CRY1

“…Amorpha-4,11-diene serves as a key and specific precursor for artemisinin biosynthesis (Bouwmeester et al 1999;Kim et al 2006). Conversion of farnesyl diphosphate to amorpha-4,11-diene, catalyzed by amorpha-4,11-diene synthase (ADS), is the first step dedicated to artemisinin biosynthesis, and cDNAs encoding this sesquiterpene cyclase have been cloned (Bouwmeester et al 1999;Kim et al 2006).…”

“…Amorpha-4,11-diene serves as a key and specific precursor for artemisinin biosynthesis (Bouwmeester et al 1999;Kim et al 2006). Conversion of farnesyl diphosphate to amorpha-4,11-diene, catalyzed by amorpha-4,11-diene synthase (ADS), is the first step dedicated to artemisinin biosynthesis, and cDNAs encoding this sesquiterpene cyclase have been cloned (Bouwmeester et al 1999;Kim et al 2006). Later, a cytochrome P450 monooxygenase encoded by a glandular trichome-specific gene, CYP71AV1, was isolated; after expression in Saccharomyces cerevisiae, this P450 enzyme was shown to catalyze the oxidation of amorpha-4,11-diene to artemisinic alcohol, artemisinic aldehyde, and artemisinic acid (Teoh et al 2006).…”

Assessment of Iranian endemic Artemisia khorassanica : karyological, genome size, and gene expressions involved in artemisinin production

“…Farnesyl diphosphate (FDP) is an important product at the branch point of terpenoid metabolism that is produced by farnesyl diphosphate synthase (FDS). The conversion of FDP into amorpha-4,11-diene, which has been proposed as the first specific precursor of artemisinin, is catalyzed by amorpha-4,11-diene synthase (ADS; Kim et al, 2006). Artemisinin, a sesquiterpene lactone, is an effective antimalarial drug, active against some cancers, hepatitis B, and schistosomiasis (Efferth, 2009) and produced in glandular trichomes of some Artemisia species, e.g., A.…”