Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L.

In plants, sesquiterpenes of different structural types are biosynthesized from the isoprenoid intermediate farnesyl diphosphate. The initial reaction of the biosynthesis is catalyzed by sesquiterpene cyclases (synthases), In Artemisia annua L. (annual wormwood), a number of such sesquiterpene cycla ses are active, We have isolated a cDNA clone encoding one of these, amorph a-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. T his clone contains a 1641-bp open reading frame coding for 546 amino acids (63.9 kDa), a 12-bp 5'-untranslated end, and a 427-bp 3'-untranslated seque nce. The deduced amino acid sequence is 32 to 51% identical with the sequen ce of other known sesquiterpene cyclases from angiosperms. When expressed i n Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosp hate. GC-MS analysis identified the olefins as (E)-beta-farnesene (0.8%), a morpha-4,11-diene (91.2%), amorpha-4,7(11)-diene (3.7%), gamma-humulene (1. 0%), beta-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the o xygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorph a-4-en-7-ol (2.1%), and alpha-bisabolol (0.3%) (tentatively). Using geranyl diphosphate as substrate, amorpha-4,11-diene synthase did not produce any monoterpenes. The recombinant enzyme has a broad pH optimum between 7.5 and 9.0 and the K-m values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 7 0, and 13 mu M, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested. (C) 2000 Academic Press.