Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

Abstract

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.

Detaljer

Författare
  • Maria Brodelius
  • Anneli Lundgren
  • Per Mercke
  • Peter E Brodelius
Enheter & grupper
Forskningsområden

Ämnesklassifikation (UKÄ) – OBLIGATORISK

  • Biologiska vetenskaper
Originalspråkengelska
Sidor (från-till)3570-3577
TidskriftEuropean Journal of Biochemistry
Volym269
Utgåva nummer14
StatusPublished - 2002
PublikationskategoriForskning
Peer review utfördJa

Relaterad forskningsoutput

Per Mercke, 2000, Per Mercke, Spolegatan 3a, 222 20 Lund. 120 s.

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

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