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 stru cture -4)GlcA-beta(1,4)-GlcNAc-alpha(1-and requires the KfiA, KfiB, KfiC, a nd 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-G lc dehydrogenase. Here, we demonstrate that KfiA is an alpha-UDP-GlcNAc gly cosyltransferase and that biosynthesis of the K5 polysaccharide involves th e concerted action of the KfiA and KfiC proteins. By site-directed mutagene sis, we determined that the acidic motif of DDD, which is conserved between the C family of glycosyltransferases, is essential for the enzymatic activ ity of KfiA III addition, by Western blot analysis, we determined that asso ciation of KfiA with the cytoplasmic membrane requires KfiC but not KfiB, w hereas the interaction of KfiC with the cytoplasmic membrane was dependent on both KfiA and KfiB. Likewise, KfiB was only detectable in cytoplasmic me mbrane fractions when both KfiA and KfiC were present. These data suggest t hat 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.