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.
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.
Hodson
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School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Griffiths
G
G
Cook
N
N
Pourhossein
M
M
Gottfridson
E
E
Lind
T
T
Lidholt
K
K
Roberts
I S
IS
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D016667:Bacterial Capsules; D004926:Escherichia coli / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; C000613611:KfiB protein, E coli; D008565:Membrane Proteins; D011135:Polysaccharides, Bacterial; C050673:capsular polysaccharide K5; C413756:KfiA protein, E coli; D017351:N-Acetylglucosaminyltransferases