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J Biol Chem, Vol. 273, Issue 19, 11752-11757, May 8, 1998

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

Gary Griffiths, Nicola J. Cook, Eva Gottfridson^§, Thomas Lind^§, Kerstin Lidholt^§, and Ian S. Roberts

From the  School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom and the ^§ Department of Medical and Physiological Chemistry, University of Uppsala, The BioMedical Center, Box 575, S-751 23 Uppsala, Sweden

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

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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