Expression, purification and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.
Abstract
A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.
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Several other bisabolanes not yet reported as natural products from A. annua were obtained from cyclization of FPP catalysed by amorpha-4,11-diene synthase.
“Several other bisabolanes, which have not yet been reported as natural products from A. annua, were also obtained from the cyclization of FPP which is catalysed by this enzyme [134]”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [134]
trans-Nerolidol and (E)-1,3(15),6,10-farnesatetraene are reported as natural products from A. annua.
“both of which are reported as natural products from A. annua [134] (Table 29).”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [134]
Amorpha-4,11-diene synthase produces small amounts of γ-humulene as well as the expected bicyclic product.
“The enzyme amorpha-4,11-diene synthase from A. annua has been observed to produce small amounts of γ-humulene (isomeric with α-humulene (401) in Table 33 – see also Figure 3, Section 3.1), as well as the expected bicyclic product [134].”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [134]
Amorpha-4,11-diene synthase also produces small amounts of acyclic sesquiterpenes trans-nerolidol and (E)-1,3(15),6,10-farnesatetraene.
“the enzyme amorpha-4,11-diene synthase, which catalyses the formation of the bicyclic sesquiterpene amorpha-4,11-diene, (Section 2.6.3) also produces small amounts of acyclic sesquiterpenes such as trans-nerolidol (380) and (E)-1,3(15),6,10-farnesatetraene (382)”
From The Biosynthesis of Artemisinin (Qinghaosu) and the Phytochemistry of Artemisia annua L. (Qinghao) (2010) · cites this paper as [134]
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References 41 · Cited by 117
- Authors
- Picaud, S., Olofsson, L., Brodelius, M., & Brodelius, P. E.
- Corresponding author
- Brodelius, Peter E.
- Venue
- Archives of Biochemistry and Biophysics
- Publisher
- Elsevier BV
- Published
- 2005-04
- Volume
- 436
- Issue
- 2
- Pages
- 215-226
- ISSN
- 0003-9861 (ISSN-L)
- OpenAlex
- W2078665241
- Cited by
- 117
- References
- 41
- Citations by year
- 2026: 1 · 2024: 10 · 2023: 5 · 2022: 2 · 2021: 4 · 2020: 1 · 2019: 4 · 2018: 2 · 2017: 9 · 2016: 10 · 2015: 3 · 2014: 7 · 2013: 11 · 2012: 6
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Archives of Biochemistry and Biophysics (publishedVersion)
- PubMed (publishedVersion)
- Funders
- European Commission
- Crafoordska Stiftelsen
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Sesquiterpenes and Asteraceae Studies
- Keywords
- amorpha-4,11-diene synthase, Artemisia annua, sesquiterpene biosynthesis, farnesyl pyrophosphate, geranyl diphosphate, enzyme purification, enzyme kinetics, metal cofactors, manganese, cobalt, magnesium, pH optimum, product selectivity, cDNA cloning
- MeSH terms
- Cations, Chromatography, Gas, Cloning, Molecular, Cobalt, Copper, Electrophoresis, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Histidine, Hydrogen-Ion Concentration, Ions, Kinetics, Magnesium, Manganese, Gas Chromatography-Mass Spectrometry, Models, Chemical, Oxygen, Phosphates, Plasmids, Recombinant Proteins, Saccharomyces cerevisiae, Substrate Specificity, Zinc, Blotting, Western, Polymerase Chain Reaction, Mutagenesis, Site-Directed, Sequence Analysis, DNA, DNA Primers, DNA, Complementary, Crystallography, X-Ray, Alkyl and Aryl Transferases, Artemisia annua
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