Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Biochemical Characterization of dTDP-Glucose 4,6-Dehydratase Gene (gerE) from Streptomyces sp. GERI-155

  • Lee, Hei-Chan (Institute of Biomolecule Reconstruction, Sun Moon University) ;
  • Sohng, Jae-Kyung (Institute of Biomolecule Reconstruction, Sun Moon University) ;
  • Kim, Hyung-Jun (Institute of Biomolecule Reconstruction, Sun Moon University) ;
  • Nam, Doo-Hyun (Department of Pharmacy, Yeungnam University) ;
  • Seong, Chi-Nam (Department of Biology, Sunchon National University) ;
  • Han, Ji-Man (Department of Pharmacy, Chosun University) ;
  • Yoo, Jin-Cheol (Department of Pharmacy, Chosun University)
  • Published : 2004.06.01
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Abstract

GERI-155 is a macrolide antibiotic containing two deoxyhexose molecules and shows antimicrobial activities against Gram-positive bacteria. Deoxysugar biosynthetic gene cluster of GERI-155 from Streptomyces sp. GERI-l55 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP g]ucose-4,6-dehydratase gene was designated as gerE. GerE was expressed in E. coli by using a recombinant expression vector pHJ1. The expressed protein was purified from E. coli cell lysate by using ammonium sulfate fractionation, and DEAE-sepharose CL-6B and hydroxylapatite column chromatography. The molecular mass of the expressed protein correlated with the predicted mass that was deduced from the cloned gene sequence data. The recombinant protein was a homodimer with a subunit relative molecular weight of 39,000 Dalton. It was found to have dTDP-glucose 4,6-dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of $K_{m} and V_{max}$ for dTDP-g]ucose were $32\mu$M and 335 nmol $min^{-1}$ (mg protein)^{-1}$, respectively. dTTP and dTDP were strong inhibitors of the protein. $NAD^+$, the coenzyme for dTDP-glucose 4,6-dehydratase, was tightly bound to the expressed protein.

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