Cyclization mechanism of amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis.
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.
Kim
Soon-Hee
SH
School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
Heo
Keon
K
Chang
Yung-Jin
YJ
Park
Si-Hyung
SH
Rhee
Sang-Ki
SK
Kim
Soo-Un
SU
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D019306:Artemisia / Q000201:enzymology*; D037621:Artemisinins / Q000378:metabolism*; D003500:Cyclization; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010946:Plants, Medicinal / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis; D012717:Sesquiterpenes / Q000378:metabolism*; D013237:Stereoisomerism
D037621:Artemisinins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; C031327:artemisinin; D003903:Deuterium; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase