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

Summary, in English

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.

• Författare: Maria Brodelius; Anneli Lundgren; Per Mercke; Peter E Brodelius
• Avdelning/ar: Kemiska institutionen
• Publiceringsår: 2002
• Språk: Engelska
• Sidor: 3570-3577
• Publikation/Tidskrift/Serie: European Journal of Biochemistry
• Volym: 269
• Issue: 14
• Länkar: http://dx.doi.org/10.1046/j.1432-1033.2002.03044.x
• Dokumenttyp: Artikel i tidskrift
• Förlag: Wiley-Blackwell
• Ämne: Biological Sciences
• Status: Published
• ISBN/ISSN/Övrigt: ISSN: 0014-2956