Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L.
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 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.
Picaud
Sarah
S
Department of Chemistry and Biomedical Sciences, University of Kalmar, 39182 Kalmar, Sweden.
Olofsson
Linda
L
Brodelius
Maria
M
Brodelius
Peter E
PE
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000302:isolation & purification*; D031172:Artemisia annua / Q000201:enzymology*; D015153:Blotting, Western; D002412:Cations; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
D002412:Cations; D017931:DNA Primers; D018076:DNA, Complementary; D007477:Ions; D010710:Phosphates; D011994:Recombinant Proteins; D003035:Cobalt; D008345:Manganese; D006639:Histidine; D003300:Copper; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase; D008274:Magnesium; D015032:Zinc; D010100:Oxygen