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KMID : 0545119990090020206
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Journal of Microbiology and Biotechnology
1999 Volume.9 No. 2 p.206 ~ p.212
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Expression of orf7(oxi III)as dTDP-Glucose 4,6-Dehydratase Gene Cloned from Streptomyces antibioticus Tii99 and Biochemical Characteristics of Expressed Protein
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Yoo, Jin Cheol
Han, Ji Man/Sohng, Jae Kyung
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Abstract
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The gene orf7(oxi Ⅲ) was expressed using an E. coli system in anticipation that it would encode dTDP-glucose 4,6-dehydratase which is involved in the biosynthesis of the olivose moiety of chlorothricin produced from Streptomyces antibioticus T?99. The solubility of the expressed protein increased up to 20% under optimal induction conditions. The expressed protein was purified from the E. coli BL 21(DE3) cell lysate by a 28.5-fold purification in two chromatography steps with a 38% recovery to near homogeneity. The molecular weight and N-terminal amino acid sequence of the purified protein correlated with the predicted mass and sequence deduced from the orf7 gene. The purified protein was a homodimer with a subunit relative molecular weight of 38,000 Dalton. The expressed protein was found to exhibit dTDP-glucose 4,6-dehydratase activity and be highly specific for dTDP-glucose as a substrate. The values of K`m and V`max for dTDP-glucose were 28㎛ and 295 nmol min^1 ㎎ protein)^1, respectively. dTTP and dTDP were strong inhibitors of this enzyme. NAD^+, the coenzyme for dTDP-glucose 4.6-dehydratase, was tightly hound to the expressed protein.
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