Journal article
Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale
Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-39182 Kalmar, Sweden.1
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.
| Language: | English |
|---|---|
| Year: | 2006 |
| Pages: | 17-28 |
| ISSN: | 00039861 and 10960384 |
| Types: | Journal article |
| DOI: | 10.1016/j.abb.2006.06.007 |
Alkyl and Aryl Transferases Amino Acid Sequence Carbon-Carbon Lyases Catalysis Cloning, Molecular DNA, Complementary Escherichia coli Gas Chromatography-Mass Spectrometry Gene Expression Regulation, Enzymologic Hydrogen-Ion Concentration Kinetics Magnesium Magnoliopsida Metals, Heavy Molecular Sequence Data Open Reading Frames Recombinant Proteins Thermodynamics Zingiber officinale germacrene D synthase trichodiene synthetase
