Characterization of the Glycosyltransferase Enzyme from theEscherichia coli K5 Capsule Gene Cluster and Identification and Characterization of the Glucuronyl Active Site

• Published in: The Journal of biological chemistry 1998-05, Vol.273 (19), p.11752-11757
• Main Authors: Griffiths, Gary, Cook, Nicola J., Gottfridson, Eva, Lind, Thomas, Lidholt, Kerstin, Roberts, Ian S.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.19.11752

Bacterial capsular polysaccharides play an important role in virulence and survival. The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-β(1,4)-GlcNAc α(1-, identical to N -acetylheparosan. A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating α and β addition of each UDP-sugar to the nonreducing end of the polysaccharide chain. Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of β-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 β addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for β-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.