Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD

Protein Expr Purif. 2002 Aug;25(3):533-40. doi: 10.1016/s1046-5928(02)00039-6.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / metabolism
  • Catalysis
  • Chromatography, Affinity
  • DNA, Complementary / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression
  • Glutamate Decarboxylase / chemistry
  • Glutamate Decarboxylase / genetics
  • Glutamate Decarboxylase / isolation & purification*
  • Glutamate Decarboxylase / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Mycobacterium tuberculosis / enzymology*
  • Protein Subunits
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment

Substances

  • DNA, Complementary
  • Protein Subunits
  • Recombinant Proteins
  • Aspartic Acid
  • aspartate-alpha-decarboxylase
  • Glutamate Decarboxylase