Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/109574
- author
- Brodelius, Maria ; Lundgren, Anneli ; Mercke, Per LU and Brodelius, Peter E
- organization
- publishing date
- 2002
- 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}}, }