PURIFICATION AND CHARACTERIZATION OF THE ESCHERICHIA-COLI K1 NEUB GENE-PRODUCT N-ACETYLNEURAMINIC ACID SYNTHETASE

Escherichia coil K1 produces a capsular polysaccharide of alpha(2-8) p oly-N-acetylneuraminic acid. This polysaccharide is an essential virul ence factor of these neuropathogenic bacteria, The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the ne uBAC genes of the K1 gene cluster, Cells harboring the neuB(+) allele in an aldolase (nanA(-)) negative background produce neuNAc in vivo, E nzymatic synthesis of neuNAc could be demonstrated in extracts of cell s harboring an expression plasmid (pNEUB) containing the neuB gene alo ne, NeuNAc synthetase was purified to homogeneity from extracts of cel ls harboring pNEUB, The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene. The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene, NeuNAc synthet ase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction, The enzyme condenses manNAc and PE P with the release of phosphate, The E,coli neuNAc synthetase is speci fic for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetyl mannosamine-6-phosphate to form neuNAc-9-PO4. This represents the firs t report of a purification of a sialic acid synthetase from either a e ukaryotic or prokaryotic source to homogeneity. These experiments clea rly demonstrate an aldolase-independent sialic acid synthetase activit y in E,coli K1.