Identification of the GDP-N-acetyl-d-perosamine Producing Enzymes From Escherichia Coli O157:H7

FEBS Lett. 2008 Feb 20;582(4):479-84. doi: 10.1016/j.febslet.2008.01.005. Epub 2008 Jan 15.

Abstract

GDP-N-acetyl-d-perosamine is a precursor of the LPS-O-antigen biosynthesis in Escherichia coli O157:H7. Like other GDP-6-deoxyhexoses, GDP-N-acetyl-d-perosamine is supposed to be synthesized via GDP-4-keto-6-deoxy-d-mannose, followed by a transamination- and an acetylation-reaction catalyzed by PerA and PerB. In this study, we have overproduced and purified PerA and PerB from E. coli O157:H7 in E. coli BL21. The recombinant proteins were partly characterized and the final product of the reaction catalyzed by PerB was shown to be GDP-N-acetyl-d-perosamine by chromatography, mass spectrometry, and 1H-NMR. The functional expression of PerB provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which is needed to further study the corresponding glycosyltransferases in vitro.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • Cloning, Molecular
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Enzymes / genetics
  • Enzymes / metabolism*
  • Escherichia coli O157 / enzymology*
  • Mannose / analogs & derivatives*
  • Mannose / biosynthesis
  • Mannose / chemistry
  • Mass Spectrometry
  • Molecular Sequence Data
  • Sequence Homology, Amino Acid

Substances

  • DNA Primers
  • Enzymes
  • 4-amino-4,6-dideoxy-D-mannose
  • Mannose