Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid. This polysaccharide is an essential virulence factor of these neuropathogenic bacteria. The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster. Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo. Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone. NeuNAc synthetase was purified to homogeneity from extracts of cells 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 synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction. The enzyme condenses manNAc and PEP with the release of phosphate. The E. coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4. This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity. These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E. coli K1.

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N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.