Expression, purification and characterization of recombinant (E)-β-farnesene synthase from Artemisia annua

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, P-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the Km- and k(cat)-values for farnesyl diphosphate, is 2.1 mu M 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 Mg2+ Mn2+ or Co2+ but essentially no activity when Zn2+, Ni2+ or Cu2+ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and < IQ mu M for Mg2+ Co2+ or Mn2+, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme. (c) 2005 Elsevier Ltd. All rights reserved.