In Vitro Characterization of a Multidomain Glycosyltransferase Using Fluorescently Tagged Synthetic Acceptors
In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-β-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or β1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.
Key wordsMicrobial glycobiology Lipopolysaccharide O antigen Glycosyltransferase In vitro biochemical reaction Fluorescently tagged synthetic acceptors
We thank Akihiko Koizumi, Iain L. Mainprize, Matthew S. Kimber, and Todd L. Lowary for their contributions to the projects originally published as refs. 2 and 3. This work was supported by the Natural Sciences and Engineering Research Council of Canada. C.W. acknowledges additional support from the Canada Research Chairs program.
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