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Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.

2005; Elsevier BV; Volume: 436; Issue: 2 Linguagem: Inglês

10.1016/j.abb.2005.02.012

1096-0384

S. Picaud, Linda Olofsson, Maria Brodelius, Peter E. Brodelius,

Microbial Natural Products and Biosynthesis

A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 μM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 × 10−3 s−1, respectively. Km and kcat for geranyl diphosphate were 16.9 μM and 7.0 × 10−4 s−1, respectively, at pH 6.5, in the presence of Mn2+.