Purification and characterization of the Escherichia coli Kl neuB gene product N-acetylneuraminic acid synthetase

Abstract

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.

Diversity of Microbial Sialic Acid Metabolism

“…However, the NeuC orthologue, SiaA (SynX), in group B meningococci is postulated to be a GlcNAc-6-P epimerase that produces ManNAc-6-P, which would then be dephosphorylated by an unknown phosphatase (96). In either case, free ManNAc is produced as the obligate substrate for the condensing (ManNAc plus phosphoenolpyruvate) enzyme, NeuB (144,152,153).…”

Discovery and characterization of sialic acid O-acetylation in group BStreptococcus

“…The genetic basis of Sia biosynthesis in GBS has been localized to a cluster of genes at the transcriptional end of the 16-gene capsule operon (32). Similar to E. coli (33,34), GBS gene products of neuC, neuB, and neuA, respectively, catalyze the epimerization of UDP-GlcNAc to N-acetyl mannosamine (ManNAc) (35), the condensation of ManNAc and phosphoenolpyruvate to form Neu5Ac (36), and the activation of Neu5Ac with CTP, to generate CMP-Neu5Ac (37). An ␣2-3 sialyltransferase encoded by cpsK then catalyzes the transfer of activated Neu5Ac to the assembling CPS (38).…”

The NeuC Protein ofEscherichia coliK1 Is a UDPN-Acetylglucosamine 2-Epimerase

“…This reaction is catalyzed by NeuB (36). Activation of NeuNAc is performed by NeuA, which adds a nucleotide monophosphate to the sugar to form CMP-NeuNAc (35,42).…”