Elongation of alternating alpha 2,8/2,9 polysialic acid by the Escherichiacoli K92 polysialyltransferase

We have chosen E. coli K92, which produces the alternating structure alpha (2-8)neuNAc alpha (2-9)neuNAc as a model system for studying bacterial poly saccharide biosynthesis. We have shown that the polysialyltransferase encod ed by the K92 neuS gene can synthesize both alpha (2-8) and alpha (2-9) neu NAc linkages in vivo by C-13-nuclear magnetic resonance analysis of polysac charide isolated from a heterologous strain containing the K92 neuS gene. T he K92 polysialyltransferase is associated with the membrane in lysates of cells harboring the neuS gene in expression vectors. Although the enzyme ca n transfer sialic acid to the nonreducing end of oligosaccharides with eith er linkage, it is unable to initiate chain synthesis without exogenously ad ded polysialic acid. Thus, the polysialyltransferase encoded by neuS is not sufficient for de novo synthesis of polysaccharide but requires another me mbrane component for initiation. The acceptor specificity of this polysialy ltransferase was studied using sialic acid oligosaccharides of various stru ctures as exogenous acceptors. The enzyme can transfer to the nonreducing e nd of all bacteria polysialic acids, but has a definite preference for alph a (2-8) acceptors. Gangliosides containing neuNAc oc(2-8)neuNAc are elongat ed, whereas monsialylated gangliosides are not. Disialylgangliosides are be tter acceptors than short oligosaccharides, suggesting a lipid-linked oligo saccharide may be preferred in the elongation reaction. These studies show that the K92 polysialyltransferase catalyzes an elongation reaction that in volves transfer of sialic acid from CMP-sialic acid to the nonreducing end of two different acceptor substrates.