Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.
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.
Griffiths
G
G
School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Cook
N J
NJ
Gottfridson
E
E
Lind
T
T
Lidholt
K
K
Roberts
I S
IS
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
D029968:Escherichia coli Proteins; D001224:Aspartic Acid; D016695:Glycosyltransferases; C097521:KfiC protein, E coli