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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Expression, Purification and Properties of Shikimate Dehydrogenase from Mycobacterium Tuberculosis

  • Zhang, Xuelian (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Zhang, Shunbao (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Hao, Fang (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Lai, Xuhui (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Yu, Haidong (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Huang, Yishu (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University) ;
  • Wang, Honghai (State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University)
  • Published : 2005.09.30
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Abstract

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is onsidered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for $\alpha$-helix, $\beta$-sheet, $\beta$-turn, and random coil were 29.2%, 9.3%, 32.7%, and 28.8%, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature $63^{\circ}C$. The apparent Michaelis constant for shikimic acid and $NADP^+$ were calculated to be about $29.5\;{\mu}M$ and $63\;{\mu}M$. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of $NADP^+$ with an enzyme turnover number of $399\;s^{-1}$. Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.

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References

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