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Originally published In Press as doi:10.1074/jbc.M004426200 on June 19, 2000

J. Biol. Chem., Vol. 275, Issue 35, 27311-27315, September 1, 2000
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Identification That KfiA, a Protein Essential for the Biosynthesis of the Escherichia coli K5 Capsular Polysaccharide, Is an alpha -UDP-GlcNAc Glycosyltransferase
THE FORMATION OF A MEMBRANE-ASSOCIATED K5 BIOSYNTHETIC COMPLEX REQUIRES KfiA, KfiB, and KfiC*

Nigel HodsonDagger , Gary GriffithsDagger , Nicola CookDagger , Meraj PourhosseinDagger §, Eva Gottfridson, Thomas Lind, Kerstin Lidholt, and Ian S. RobertsDagger ||

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

The Escherichia coli K5 capsular polysaccharide consists of the repeat structure -4)GlcA-beta (1,4)-GlcNAc-alpha (1- and requires the KfiA, KfiB, KfiC, and KfiD proteins for its synthesis. Previously, the KfiC protein was shown to be a beta -UDP-GlcA glycosyltransferase, and KfiD was shown to be a UDP-Glc dehydrogenase. Here, we demonstrate that KfiA is an alpha -UDP-GlcNAc glycosyltransferase and that biosynthesis of the K5 polysaccharide involves the concerted action of the KfiA and KfiC proteins. By site-directed mutagenesis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activity of KfiA. In addition, by Western blot analysis, we determined that association of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, whereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic membrane fractions when both KfiA and KfiC were present. These data suggest that the interaction between the KfiA, KfiB, and KfiC proteins is essential for the stable association of these proteins with the cytoplasmic membrane and the biosynthesis of the K5 polysaccharide.

* The work in the laboratory of I. S. R. was supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom, the Wellcome Trust, and the Lister Institute of Preventive Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a studentship from the Iranian Ministry of Health and Medical Education.

|| To whom correspondence should be addressed. Tel.: 0161-275-5601; Fax: 0161-275-5656; E-mail: ISRobert@fs1.scg.man.ac.uk.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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