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Molecular Cloning, Expression, and Characterization of Amorpha-4,11-diene Synthase, a Key Enzyme of Artemisinin Biosynthesis in Artemisia annua L.
Open accessAbstract
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)-beta-farnesene (0.8%), amorpha-4,11diene (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 oxygenated sesquiterpenes as amorpha-4-en-11-ol (0.2%) (tentatively), amorpha-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 Km values for farnesyl diphosphate, Mg2+, and Mn2+ are 0.9, 70, and 13 microM, respectively, at pH 7.5. A putative reaction mechanism for amorpha-4,11-diene synthase is suggested.
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Metadata
- Authors
- Mercke, P., Bengtsson, M., Bouwmeester, H. J., Posthumus, M. A., & Brodelius, P. E.
- Affiliations
- Lund University (SE)
- Swedish University of Agricultural Sciences (SE)
- Wageningen University & Research (NL)
- Corresponding author
- Brodelius, Peter E.
- Venue
- Archives of Biochemistry and Biophysics
- Publisher
- Elsevier BV
- Published
- 2000-09
- Volume
- 381
- Issue
- 2
- Pages
- 173-180
- ISSN
- 0003-9861 (ISSN-L)
- OpenAlex
- W2023533798
- Cited by
- 283
- References
- 27
- Citations by year
- 2026: 7 · 2025: 8 · 2024: 13 · 2023: 10 · 2022: 10 · 2021: 7 · 2020: 5 · 2019: 13 · 2018: 13 · 2017: 16 · 2016: 21 · 2015: 10 · 2014: 11 · 2013: 13 · 2012: 17
- FWCI
- 1.56
- Type
- journal article
- Language
- en
- Open access
- green
- research.wur.nl
- Locations
- Archives of Biochemistry and Biophysics (publishedVersion)
- PubMed (publishedVersion)
- Socio-Environmental Systems Modeling (acceptedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Funders
- Skogs- och Jordbrukets Forskningsråd
- Vetenskapsrådet
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Physical Sciences › Chemistry › Organic Chemistry › Sesquiterpenes and Asteraceae Studies
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant tissue culture and regeneration
- Keywords
- artemisinin biosynthesis, Artemisia annua, sesquiterpene cyclase, farnesyl pyrophosphate, sesquiterpene biosynthesis, cDNA cloning, recombinant enzyme, enzyme kinetics, enzyme mechanism
- MeSH terms
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Escherichia coli, Kinetics, Models, Chemical, Molecular Sequence Data, Plants, Medicinal, Polyisoprenyl Phosphates, Sesquiterpenes, Gene Expression, Genes, Plant, Sequence Homology, Amino Acid, DNA Primers, DNA, Complementary, DNA, Plant, Artemisia, Alkyl and Aryl Transferases, Artemisinins
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