title: Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd.
abstract: The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K(m) for UDP-GalNAc and globotriose are 58 microM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Shao
authors.forename: Jianbo
authors.initials: J
authors.lastname: Zhang
authors.forename: Przemyslaw
authors.initials: P
authors.lastname: Kowal
authors.forename: Yuquan
authors.initials: Y
authors.lastname: Lu
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D014312:Trisaccharides / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; C113460:globotriose; C462020:LgtD protein, Haemophilus influenzae; D017350:N-Acetylgalactosaminyltransferases