Cited by 285Plant biochemistry and biosynthesisSesquiterpenes and Asteraceae StudiesPlant tissue culture and regeneration

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

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Mercke, Per, Bengtsson, Marie, Bouwmeester, Harro J., Posthumus, Maarten A., Brodelius, Peter E.·2000·Archives of Biochemistry and Biophysics

Abstract

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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Crossref · 10.1006/abbi.2000.1962

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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
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
  • 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
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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