Identification of the GDP-N-acetyl-d-perosamine producing enzymes from Escherichia coli O157:H7.
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.
Albermann
Christoph
C
Institute of Microbiology, Universität Stuttgart, Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. imbca@imb.unni-stuttgart.de
Beuttler
Holger
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D019453:Escherichia coli O157 / Q000201:enzymology*; D008358:Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004798:Enzymes; C057033:4-amino-4,6-dideoxy-D-mannose; D008358:Mannose