Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.
A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.
Picaud
Sarah
S
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182 Kalmar, Sweden.
Olsson
Mikael E
ME
Brodelius
Maria
M
Brodelius
Peter E
PE
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D015971:Gene Expression Regulation, Enzymologic; D020939:Zingiber officinale / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019684:Magnoliopsida / Q000201:enzymology; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D018076:DNA, Complementary; D019216:Metals, Heavy; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C470416:germacrene D synthase; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D008274:Magnesium