Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD.
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.
Chopra
Sidharth
S
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, ICGEB Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Pai
Harish
H
Ranganathan
Anand
A
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D001224:Aspartic Acid; C019104:aspartate-alpha-decarboxylase; D005968:Glutamate Decarboxylase