title: Functional identification of bacterial glucosyltransferase WbdN.
abstract: The outer membrane of gram-negative bacteria is stabilized by lipopolysaccharides (LPS). The O-antigenic polysaccharides of LPS are composed of repeating units that are exposed to and can interact with the environment. The glycosyltransferases that assemble these repeating units are encoded by the O-antigen gene cluster and utilize undecaprenol-phosphate-linked intermediates as natural acceptor substrates, and nucleotide sugars as donor substrates on the cytoplasmic face of the inner membrane. Many of the glycosyltransferase genes are known but the enzymatic functions of most of them remain to be identified. We describe here how the function of a recombinant glucosyltransferase WbdN from Escherichia coli O157 can be determined by NMR analysis of the enzyme product, using a synthetic acceptor substrate analog. A fluorescent acceptor substrate analog can be used in highly sensitive enzyme assays that allow the characterization of enzyme activity without the use of radioactive nucleotide sugar donor substrates.
authors.affiliation: Department of Medicine, Division of Rheumatology, Queen's University, Kingston, Ontario, Canada.
authors.forename: Yin
authors.initials: Y
authors.lastname: Gao
authors.forename: Anna
authors.initials: A
authors.lastname: Vinnikova
authors.forename: Inka
authors.initials: I
authors.lastname: Brockhausen
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D019453:Escherichia coli O157 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005453:Fluorescence; D005964:Glucosyltransferases / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C585531:WbdN protein, E coli