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KMID : 0545120040140030576
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 3 p.576 ~ p.583
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Cloning, Expression, and Biochemical Characterization of dTDP-Glucose 4,6-Dehydratase Gene (gerE) from Streptomyces sp. GERI-155
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Lee HC
Sohng JK/Kim HJ/Nam DH/Seong CN/Han JM/Yoo JC
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Abstract
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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-155 genome was cloned. Four orfs were identified and a putative orf presumed to be the dTDP glucose-46-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 39000 Dalton. It was found to have dTDP-glucose 46- dehydratase activity and also found to be highly specific for dTDP-glucose as a substrate. The values of Km and Vmax for dTDP-glucose were 32 mM and 335 nmol min-1 (mg protein)-1 respectively. dTTP and dTDP were strong inhibitors of the protein. NAD+ the coenzyme for dTDP-glucose 46-dehydratase was tightly bound to the expressed protein.
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