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Journal of Bacteriology, February 2004, p. 706-712, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.706-712.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase

Willie F. Vann,1* Dayle A. Daines,2,{dagger} Andrew S. Murkin,3 Martin E. Tanner,3 Donald O. Chaffin,4 Craig E. Rubens,4 Justine Vionnet,1 and Richard P. Silver2

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York 14642,2 Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892,1 Department of Chemistry, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1,3 Department of Pediatrics, University of Washington, Children's Hospital and Regional Medical Center, Seattle, Washington 981054

Received 18 July 2003/ Accepted 27 October 2003

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 ({Delta}neuC), 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 {Delta}neuC. 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 [2-14C]acetamidoglucal and [N-14C]acetylmannosamine (ManNAc) from UDP-[14C]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.

* Corresponding author. Mailing address: Laboratory of Bacterial Toxins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. Phone: (301) 496-2008. Fax: (301) 402-2776. E-mail: wvann{at}helix.nih.gov.

{dagger} Present address: Seattle Biomedical Research Institute, Seattle, WA 98109-1651.

Journal of Bacteriology, February 2004, p. 706-712, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.706-712.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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