Characterization of mycobacterial UDP-N-acetylglucosamine enolpyruvyle transferase (MurA)

Res Microbiol. 2014 Feb-Mar;165(2):91-101. doi: 10.1016/j.resmic.2014.01.004. Epub 2014 Jan 23.

Abstract

The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs.

Keywords: Cell wall; MurA; Mycobacteria; Peptidoglycan; UDP-N-acetylglucosamine enolpyruvyle transferase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkyl and Aryl Transferases / genetics*
  • Alkyl and Aryl Transferases / metabolism*
  • Cloning, Molecular
  • Colorimetry / methods
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression
  • Gene Knockout Techniques
  • Genes, Essential*
  • Kinetics
  • Microbial Viability / radiation effects
  • Microscopy, Electron, Scanning
  • Mycobacterium smegmatis / enzymology*
  • Mycobacterium smegmatis / genetics
  • Mycobacterium smegmatis / ultrastructure
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Temperature

Substances

  • Recombinant Proteins
  • Alkyl and Aryl Transferases
  • UDP-N-acetylglucosamine 1-carboxyvinyltransferase