tailieunhanh - Báo cáo Y học: Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

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, . FPPS/ eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-SerGly) 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. | Eur. J. Biochem. 269 3570-3577 2002 FEBS 2002 doi Fusion of farnesyldiphosphate synthase and epZ-aristolochene synthase a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum Maria Brodelius1 Anneli Lundgren1 Per Mercke2 t and Peter E. Brodelius1 1 Department of Chemistry and Biomedical Sciences University of Kalmar Sweden department of Plant Biochemistry Lund University Sweden 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 . 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-b-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. Keywords bifunctional enzyme epi-aristolochene synthase farnesyl diphosphate synthase gene fusion recombinant expression. The enzymatic machinery of a living cell is very complex. Thousands of enzymes are present and the flow of metabolites has to be tightly regulated. .

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