The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase - PubMed (original) (raw)

The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase

Willie F Vann et al. J Bacteriol. 2004 Feb.

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.

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Figures

FIG. 1.

FIG. 1.

Polyacrylamide gel-purified:NeuC. The mobilities of molecular weight (M.W.) standards (Std.) are shown at the right.

FIG. 2.

FIG. 2.

UDP-GlcNAc 2-epimerase activity of NeuC. (A) Autoradiogram. Purified enzyme was incubated with UDP-[14C]GlcNAc for 3 h at 37°C (lane 1). As a control, enzyme was boiled for 5 min prior to the addition of substrate (lane 2). A total of 40 μl of each reaction mixture was spotted at the origin of borate-impregnated paper, and the chromatogram was developed in ethyl acetate-2-propanol-pyridine-H2O (50:22:14:14). (B) Sugar standard. One milligram of each sugar per milliliter was spotted (25 μl), and the chromatogram was developed with silver nitrate.

FIG. 3.

FIG. 3.

(A and B) 1H NMR spectra of the incubation of UDP-GlcNAc with NeuC after 3 min (A) and 43.5 h (B). t, time. (C) 1H NMR spectrum of an authentic sample of 2-acetamidoglucal. The position of the anomeric proton is indicated by the arrow.

FIG. 4.

FIG. 4.

(A and B) 31P NMR spectra of the incubation of UDP-GlcNAc with NeuC after 6 min (A) and 43.5 h (B). t, time. (C) 31P NMR spectrum of an authentic sample of UDP.

FIG. 5.

FIG. 5.

Proposed mechanism of the reaction catalyzed by the mammalian UDP-GlcNAc 2-epimerase.

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