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Sesquiterpene synthases in Artemisia annua: Cloning, Expression, and Characterisation of the Recombinant Enzymes

Author

Summary, in English

Plants produce a multitude of different compounds that may have biotic effects. Artemisia annua, produces a compound used as a drug against multi-drug-resistant strains of Plasmodium falciparum, the parasite causing malaria. The compound, artemisinin, is a sesquiterpene and is an essential component for the production of artemisinin drugs against malaria. In a first critical step of the biosynthesis of artemisinin, a sesquiterpene carbon structure is formed from the substrate farnesyl diphosphate. Enzymes catalysing sesquiterpene formation, are called sesquiterpene synthases. The common substrate farnesyl diphosphate may, enzymatically, be converted to over 300 different sesquiterpene hydrocarbons. Sesquiterpene synthases are thought to be regulatory enzymes for the intracellular carbon metabolic flux in plants. Epi-cedrol synthase and amorpha-4,11-diene synthase have been cloned from Artemisia annua. The cDNA for these clones have been expressed in E. coli and the recombinant enzymes have been identified and characterised. Recombinant epi-cedrol synthase catalyses the formation of both olefinic (3%) and oxygenated (97%) products from farnesyl diphosphate. The major product formed by this enzyme is 8-epi-cedrol. The other recombinant enzyme catalyses the synthesis of amorpha-4,11-diene, as a major product. Amorpha-4,11-diene has the correct structure to be the precursor for artemisinin. A reaction mechanism starting with a C1,C6 closure is proposed for both recombinant terpene synthases. Both recombinant enzymes demonstrate kinetic characteristics comparable to other cloned sesquiterpene synthases. Fusing the open reading frames of farnesyl diphospahte synthase and epi-aristolochene synthase (a sesquiterpene synthase from tobacco) in different orders resulted in two bifunctional enzymes. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (G-S-G) to introduce a linker between the two open reading frames. A channeling of the intermediate farnesyl diphosphate by the bifunctional enzymes was established.

Publishing year

2000

Language

English

Document type

Dissertation

Publisher

Per Mercke, Spolegatan 3a, 222 20 Lund

Topic

  • Biological Sciences

Keywords

  • Artemisia annua
  • Sesquiterpene synthases
  • artemisinin
  • Plant biochemistry
  • Växtbiokemi

Status

Published

Supervisor

  • [unknown] [unknown]

ISBN/ISSN/Other

  • ISBN: 91-973252-8-7
  • ISRN: LUNKDL/NK VK-00/1019

Defence date

1 January 2000

Defence time

10:15

Defence place

Room 213, dep of Plant Biochemistry

Opponent

  • Joe Chappell (Prof)