title: Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.
abstract: Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein. Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein. The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity. A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein. This protein lacked 139 amino acids at the C terminus. This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.
authors.affiliation: School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
authors.forename: G
authors.initials: G
authors.lastname: Griffiths
authors.forename: N J
authors.initials: NJ
authors.lastname: Cook
authors.forename: E
authors.initials: E
authors.lastname: Gottfridson
authors.forename: T
authors.initials: T
authors.lastname: Lind
authors.forename: K
authors.initials: K
authors.lastname: Lidholt
authors.forename: I S
authors.initials: IS
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D016667:Bacterial Capsules / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D016695:Glycosyltransferases / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D012995:Solubility; D013329:Structure-Activity Relationship
chemical_list: D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D016695:Glycosyltransferases; C097521:KfiC protein, E coli