Role of undecaprenyl phosphate in synthesis of polymers containing sialic acid in Escherichia coli.

Membrane-associated sialytransferase complexes from Escherichia coli K-235 catalyze the incorporation of N-acetylneuraminic acid (NeuNAc) from cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NeuNAc) into polymeric products and a lipid fraction. Reconstitution of enzyme activity in lipid-depleted membrane complexes had an absolute dependence on a purified phospholipid which was characterized by high resolution mass spectrometry following dephosphorylation as undecaprenol. An identical mass spectrum was obtained on the phosphorylated lipid confirming that the active derivative was undecaprenyl phosphate. This C55-isoprenoid alcohol accounted for 95% of the polyisoprenol and contained 11 isoprene units, each one unsaturated. The remaining 5% was composed of the C60 homologue, dodecaprenol. Ficarprenyl phosphate, an isomer of undecaprenyl phosphate, also restored enzymatic activity although on a molar basis, it was less active than undecaprenyl phosphate. These results provide direct evidence that sialyl polymer synthesis has an obligatory requirement for undecaprenyl phosphate, a membrane-bound lipid coenzyme which functions as an intermediate carrier of glycosyl residues in the biosynthesis of a variety of microbial cell surface polysaccharides. These data extend further the general class of polysaccharides whose synthesis involves undecaprenyl phosphate and are in accord with the hypothesis that this lipid acts as an intermediate carrier of sialyl residues in sialyl polymer synthesis according to the following reaction: CMP-NeuNAc plus P-undecaprenol in equilibrium NeuNAc-P-undecaprenol plus CMP Although the sialylated lipid remains to be characterized, evidence in support of this conclusion was obtained by kinetic analysis of N-acetylneuraminic acid transfer into the lipid soluble fraction and sialyl polymers. These studies showed a rapid incorporation of N-acetylneuraminic acid into the lipid-soluble fraction prior to maximal sialyl polymer formation, an observation consistent with a possible precursortion, an observation consistent with a possible precursor-product relationship. Confirmation that the radioactivity in the lipid-soluble fraction was lipid-linked N-acetylneuraminic acid was provided by the demonstration of the formation of a transitory sialyl-lipid with chromatographic properties expected of sialyl-undecaprenyl phosphate. Three additional lines of investigation implicated a functional role for undecaprenyl phosphate in sialyl polymer synthesis...