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.
Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, ICGEB Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Sidharth
S
Chopra
Harish
H
Pai
Anand
A
Ranganathan
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