Expression,purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase,PanD |
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Authors: | Chopra Sidharth Pai Harish Ranganathan Anand |
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Institution: | Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, ICGEB Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India. |
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Abstract: | Like all bacteria, Mycobacterium tuberculosis (Mtb) possesses the genes necessary for coenzyme A biosynthesis and metabolism. In the present work, the Mtb panD gene was PCR amplified, overexpressed, and purified by metal affinity chromatography. The recombinant Mtb panD was found to exist as a tetramer in solution. Incubation of Mtb panD at 37 degrees C for several hours resulted in a complete cleavage of the inactive (pi) form into the two subunits (alpha and beta). The cleavage was confirmed by Western blot analysis as well as by N-terminal sequencing. Cleaved Mtb panD was assayed for decarboxylase activity with L-aspartate as substrate. The kinetic parameters K(m) and k(cat) were found to be 219 microM and 0.65s(-1), respectively. These results provide the means for further studies based on the identification of the Mtb panD as well as other components of pantothenate metabolism as potential drug targets. |
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