Journal Article

Purification and characterization of the <i>Escherichia coli</i> Kl <i>neu</i>B gene product N-acetylneuraminic acid synthetase

Willie F. Vann, Jose J. Tavarez, Jane Crowley, Eric Vimr and Richard P. Silver

in Glycobiology

Published on behalf of Society for Glycobiology

Volume 7, issue 5, pages 697-701
Published in print July 1997 | ISSN: 0959-6658
Published online July 1997 | e-ISSN: 1460-2423 | DOI:
Purification and characterization of the Escherichia coli Kl neuB gene product N-acetylneuraminic acid synthetase

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Escherichia coli K1 produces a capsular polysaccharide of α(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-acetylman-nosamine-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 al-dolase-independent sialic acid synthetase activity in E.coli K1.

Keywords: sialic acid synthetase; E. coli neuB; enzyme purification; polysialic acid; sialic acid metabolism

Journal Article.  0 words. 

Subjects: Carbohydrates

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