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

Brodelius, Maria ; Lundgren, Anneli ; Mercke, Per LU and Brodelius, Peter E (2002) In European Journal of Biochemistry 269(14). p.3570-3577
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... (More)
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. (Less)
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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
European Journal of Biochemistry
volume
269
issue
14
pages
3570 - 3577
publisher
Wiley-Blackwell
external identifiers
  • wos:000176920600026
  • pmid:12135497
  • scopus:0036373903
ISSN
0014-2956
DOI
10.1046/j.1432-1033.2002.03044.x
language
English
LU publication?
yes
id
76337ac6-36e6-4fff-b6e8-dfb8093c0fd5 (old id 109574)
date added to LUP
2016-04-01 16:24:59
date last changed
2022-03-30 07:42:05
@article{76337ac6-36e6-4fff-b6e8-dfb8093c0fd5,
  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.}},
  author       = {{Brodelius, Maria and Lundgren, Anneli and Mercke, Per and Brodelius, Peter E}},
  issn         = {{0014-2956}},
  language     = {{eng}},
  number       = {{14}},
  pages        = {{3570--3577}},
  publisher    = {{Wiley-Blackwell}},
  series       = {{European Journal of Biochemistry}},
  title        = {{Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.}},
  url          = {{http://dx.doi.org/10.1046/j.1432-1033.2002.03044.x}},
  doi          = {{10.1046/j.1432-1033.2002.03044.x}},
  volume       = {{269}},
  year         = {{2002}},
}