Cloning, Expression, and Characterization of epi-Cedrol Synthase, a Sesquiterpene Cyclase from Artemisia annua L
Abstract
Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-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% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) 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 substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.
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Five different sesquiterpene synthases (ECS, ADS, CPS, GAS, and BFS) have been cloned from A. annua.
“so far five different sesquiterpene synthases (epi-cedrol synthase (ECS) [5], amorpha-4,11-diene synthase (ADS) [6]; β-caryophyllene synthase (CPS) [7], germacrene A synthase (GAS) [8] and β-farnesene synthase (BFS) [9] have been cloned from the plant.”
From Relative expression of genes of terpene metabolism in different tissues of Artemisia annuaL (2011) · cites this paper as [5]
A cDNA clone encoding epi-cedrol synthase catalyzes the formation of epi-cedrol from FPP (378).
“A cDNA clone encoding epi-cedrol synthase has been shown to catalyze the formation of the oxygenated sesquiterpene epi-cedrol from FPP (378) [258,259].”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [258]
Trace amounts of α-cedrene, β-cedrene, (E)-β-farnesene, α-acoradiene and (E)-β-bisabolene were also produced by the operation of epi-cedrol synthase.
“Trace amounts of the sesquiterpene hydrocarbons α-cedrene, β-cedrene, (E)-β-farnesene, α-acoradiene and (E)-β-bisabolene were also produced by the operation of this enzyme [258].”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [258]
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- Authors
- Mercke, P., Crock, J., Croteau, R., & Brodelius, P. E.
- Affiliations
- Lund University (SE)
- Washington State University (US)
- Corresponding author
- Brodelius, Peter E.
- ORCID
- Brodelius, Peter E.
- Venue
- Archives of Biochemistry and Biophysics
- Publisher
- Elsevier BV
- Published
- 1999-09
- Volume
- 369
- Issue
- 2
- Pages
- 213-222
- ISSN
- 0003-9861 (ISSN-L)
- OpenAlex
- W1967231259
- Cited by
- 127
- References
- 33
- Citations by year
- 2025: 2 · 2024: 8 · 2023: 3 · 2022: 1 · 2021: 3 · 2019: 4 · 2018: 3 · 2017: 6 · 2016: 6 · 2015: 8 · 2014: 2 · 2013: 7 · 2012: 5
- FWCI
- 1.38
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Archives of Biochemistry and Biophysics (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM31354)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Sesquiterpenes and Asteraceae Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
- Keywords
- sesquiterpene cyclase, Artemisia annua, cedrol, sesquiterpenes, farnesyl pyrophosphate, geranylgeranyl diphosphate, α-cedrene, terpinene, terpinolene, limonene, enzyme characterization
- MeSH terms
- Amino Acid Sequence, Antimalarials, Cloning, Molecular, DNA, Hydrogen-Ion Concentration, Models, Chemical, Molecular Sequence Data, Oils, Volatile, Plants, Medicinal, Polyisoprenyl Phosphates, Recombinant Proteins, Sesquiterpenes, Substrate Specificity, Sequence Homology, Amino Acid, RNA, Plant, Asteraceae, Carbon-Carbon Lyases, Artemisinins
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