Assay for a Galactosyltransferase Involved in the Assembly of the O7-Antigen Repeat Unit of Escherichia coli
Gram-negative bacteria have lipopolysaccharides terminating in repeating oligosaccharides which comprise the O-antigen. The glycosyltransferases (GTs) assembling the O-chain utilize lipid-linked acceptor substrates and nucleotide sugar donor substrates. The natural undecaprenol-linked acceptor substrates are not readily available, precluding the characterization of O-chain GTs. This chapter describes an assay for a galactosyltransferase (GalT) involved in the synthesis of the O7 antigen of Escherichia coli VW187. The glycolipid GlcNAcα-pyrophosphate bound to a phenoxyundecyl moiety, which resembles the natural substrate, was synthesized and employed in GT assays using bacterial membranes as the enzyme source. The assay is simple and allows optimization and characterization of the enzyme reaction. Similar protocols can be used to assay other GTs in the O-chain biosynthesis pathways.
KeywordsEnzyme Product Flash Chromatography Acceptor Substrate Benzyl Bromide Pyridinium Salt