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Fusion of farnesyld...
Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.
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Brodelius, Maria (författare)
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Lundgren, Anneli (författare)
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- Mercke, Per (författare)
- Lund University,Lunds universitet,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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Brodelius, Peter E (författare)
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(creator_code:org_t)
- 2002-07-11
- 2002
- Engelska.
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:14, s. 3570-3577
- Relaterad länk:
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http://dx.doi.org/10...
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https://febs.onlinel...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Publikations- och innehållstyp
- art (ämneskategori)
- ref (ämneskategori)
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