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Characterization of Gla_KP, a UDP-Galacturonic Acid C4-Epimerase from Klebsiella pneumoniae with Extended Substrate Specificity

Department of Molecular and Cellular Microbiology, University of Guelph, Guelph Ontario, N1G 2W1, Canada

Received 25 January 2005/ Accepted 10 March 2005

In Escherichia coli and Salmonella enterica, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast, Klebsiella pneumoniae lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. Gla_KP is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of Gla_KP, and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the K_m for UDP-glucuronic acid of 13.0 µM. Gla_KP exists as a dimer in its native form. NAD⁺/NADH is tightly bound by the enzyme and addition of supplementary NAD⁺ is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. Gla_KP was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded gla_KP partially complemented a galE mutation in S. enterica and in K. pneumoniae; however, chromosomal gla_KP could not substitute for galE in a K. pneumoniae galE mutant in vivo.

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