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, 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.
Brodelius
Maria
M
Department of Chemistry and Biomedical Sciences, University of Kalmar, Sweden.
Lundgren
Anneli
A
Mercke
Per
P
Brodelius
Peter E
PE
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019306:Artemisia / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli; D005813:Genes, Synthetic; D051228:Geranyltranstransferase; D045782:Hemiterpenes; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D016366:Open Reading Frames / Q000235:genetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012717:Sesquiterpenes / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology*
D018076:DNA, Complementary; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; C081843:capsidiol; C054277:aristolochene; C004809:isopentenyl pyrophosphate; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D007535:Isomerases; C060050:aristolochene synthase