Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase.
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.
Vann
W F
WF
Laboratory of Bacterial Polysaccharides, Center for Biologics Research and Review, Bethesda, MD 20892, USA.
Tavarez
J J
JJ
Crowley
J
J
Vimr
E
E
Silver
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D005810:Multigene Family; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D013379:Substrate Specificity; D013816:Thermodynamics
D010446:Peptide Fragments; D017886:Durapatite; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases