The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua*
- Yansheng Zhang‡,
- Keat H. Teoh‡,1,
- Darwin W. Reed‡,
- Lies Maes§,
- Alain Goossens§,
- Douglas J. H. Olson‡,
- Andrew R. S. Ross‡,2 and
- Patrick S. Covello‡,3
- ‡Plant Biotechnology Institute, Saskatoon, Saskatchewan S7N OW9, Canada and the §Department of Plant Systems Biology, Flanders Institute for Biotechnology and the Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium
- ↵3 To whom correspondence should be addressed: Plant Biotechnology Institute, 110 Gymnasium Pl., Saskatoon, Saskatchewan S7N 0W9, Canada. Tel.: 306-975-5269; Fax: 306-975-4839; E-mail: patrick.covello{at}nrc-cnrc.gc.ca.
Abstract
At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Δ11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Δ11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small α,β-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.
Footnotes
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↵* This work was supported by the National Research Council of Canada's Crop for Enhanced Human Health Program, Ghent University Project VARL9104, and Vlaamse Interuniversitaire Raad Grant VLADOC-B/09269/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental data, supplemental Table S1, and supplemental Figs. S1–S4.
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↵1 Present address: Arkansas Bioscience Institute, Arkansas State University, P. O. Box 639, State University, AR 72467.
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↵2 Present address: Institute of Ocean Sciences, 9860 W. Saanich Rd., Sidney, British Columbia V8L 4B2, Canada.
- Received April 22, 2008.
- Revision received May 12, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
