Molecular Cloning, Expression, and Characterization of Amorpha-4,11-diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L

• Published in: Archives of biochemistry and biophysics 2000-09, Vol.381 (2), p.173-180
• Main Authors: Mercke, Per, Bengtsson, Marie, Bouwmeester, Harro J., Posthumus, Maarten A., Brodelius, Peter E.
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - metabolism; Amino Acid Sequence; amorpha-4,11-diene synthase; Artemisia - enzymology; Artemisia - genetics; Artemisia annua; Artemisinins; bacterial expression; Base Sequence; cDNA cloning; Cloning, Molecular; DNA Primers - genetics; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; DNA, Plant - genetics; DNA, Plant - isolation & purification; Escherichia coli - genetics; GC–MS; Gene Expression; Genes, Plant; Kinetics; Laboratorium voor Organische chemie; Laboratory for Organic Chemistry; Models, Chemical; Molecular Sequence Data; Organic Chemistry; Organische Chemie; Plant Research International; Plants, Medicinal; Polyisoprenyl Phosphates - metabolism; Sequence Homology, Amino Acid; Sesquiterpenes - chemistry; Sesquiterpenes - metabolism; sesquiterpenes monoterpenes; VLAG
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2000.1962

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 cyclases are active. We have isolated a cDNA clone encoding one of these, amorpha-4,11-diene synthase, a putative key enzyme of artemisinin biosynthesis. This 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 sequence. The deduced amino acid sequence is 32 to 51% identical with the sequence of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (97.5%) and oxygenated (2.5%) sesquiterpenes from farnesyl diphosphate. GC–MS analysis identified the olefins as (E)-β-farnesene (0.8%), amorpha-4,11-diene (91.2%), amorpha-4,7(11)-diene (3.7%), γ-humulene (1.0%), β-sesquiphellandrene (0.5%), and an unknown olefin (0.2%) and the oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-4-en-7-ol (2.1%), and α-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 Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 μM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.