Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.
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Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.
Arch Biochem Biophys. 2006 Jun 21;
Authors: Picaud S, Olsson ME, Brodelius M, Brodelius PE
A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA () 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 Mg(2+), Mn(2+), Ni(2+) or Co(2+), while the enzyme is inactive when Cu(2+) or Zn(2+) is used. The K(m)- and k(cat)-values are 0.88muM and 3.34×10(-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.
PMID: 16839518 [PubMed - as supplied by publisher]
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