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Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site.
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1998 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 19, 11752-7 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
1998. Vol. 273, no 19, 11752-7 p.
URN: urn:nbn:se:uu:diva-133866PubMedID: 9565598OAI: oai:DiVA.org:uu-133866DiVA: diva2:370470
Available from: 2010-11-16 Created: 2010-11-16 Last updated: 2011-05-17

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Lidholt, K
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