Identification of the GDP-N-acetyl-d-perosamine producing enzymes from Escherichia coli O157:H7

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.

Abbreviations: CoA, coenzyme A, GDP, guanosine diphosphate, Gmd, GDP-mannose-4,6-dehydratase, HPLC, high performance liquid chromatography, NDP, nucleotide diphosphate, NMR, nuclear magnetic resonance, PCR, polymerase chain reaction, TLC, thin layer chromatography

Keywords: GDP-N-acetyl-d-perosamine, LPS-O-antigen biosynthesis, Escherichia coli O157:H7