Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L

Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoi d intermediate farnesyl diphosphate to various sesquiterpene structural typ es. In plants, many sesquiterpenes are produced as defensive chemicals (phy toalexins) or mediators of chemical communication (i.e., pollinator attract ants). A number of sesquiterpene synthases are present in Artemisia annua L . (annual wormwood). We have isolated a cDNA clone encoding one of these, e pi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% ide ntical with the sequences of other known sesquiterpene cyclases from angios perms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes fr om farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-ced rene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alp ha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (1 6%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated , exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as subs trate, whereas geranyl diphosphate was converted to monoterpenes by the rec ombinant enzyme at a rate of about 15% of that observed with farnesyl dipho sphate as substrate. The monoterpene olefin products are limonene (45%), te rpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2 %); a small amount of the monoterpene alcohol terpinen-4-ol is also produce d. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diph osphate as substrate) and the IT, values for farnesyl diphosphate are 0.4 a nd 1.3 mu M at pH 7.0 and 9.0, respectively. The K-m for Mg2+ is 80 mu M at pH 7.0 and 9.0. (C) 1999 Academic Press.