title: Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua.
abstract: A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-beta-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI=5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, beta-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K(m)- and k(cat)-values for farnesyl diphosphate, is 2.1 microM and 9.5 x 10(-3) s(-1), respectively resulting in the efficiency 4.5 x 10(-3) M(-1)s(-1). The enzyme exhibits substantial activity in the presence of Mg(2+), Mn(2+) or Co(2+) but essentially no activity when Zn(2+), Ni(2+) or Cu(2+) is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 microM for Mg(2+), Co(2+) or Mn(2+), respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.
authors.affiliation: Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182, Kalmar, Sweden.
authors.forename: Sarah
authors.initials: S
authors.lastname: Picaud
authors.forename: Maria
authors.initials: M
authors.lastname: Brodelius
authors.forename: Peter E
authors.initials: PE
authors.lastname: Brodelius
mesh_terms: D031172:Artemisia annua / Q000201:enzymology* / Q000378:metabolism; D002412:Cations; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002412:Cations; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C109459:beta-farnesene synthase