title: The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase.
abstract: The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642, USA. wvann@helix.nih.gov
authors.forename: Willie F
authors.initials: WF
authors.lastname: Vann
authors.forename: Dayle A
authors.initials: DA
authors.lastname: Daines
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Murkin
authors.forename: Martin E
authors.initials: ME
authors.lastname: Tanner
authors.forename: Donald O
authors.initials: DO
authors.lastname: Chaffin
authors.forename: Craig E
authors.initials: CE
authors.lastname: Rubens
authors.forename: Justine
authors.initials: J
authors.lastname: Vionnet
authors.forename: Richard P
authors.initials: RP
authors.lastname: Silver
mesh_terms: D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy
chemical_list: D029968:Escherichia coli Proteins; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase