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J. Biol. Chem., Vol. 279, Issue 36, 37551-37558, September 3, 2004

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Biochemical Characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1^*

Wayne L. Miller, Cory Q. Wenzel, Craig Daniels¶, Suzon Larocque||, Jean-Robert Brisson||, and Joseph S. Lam**

From the Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and the ^||Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-D-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD⁺ as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-D-glucosaminuronic acid. WbpA requires either NH₄⁺ or K⁺ for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-D-glucosamine binding with a K_0.5 of 94 µM, a k_cat of 86 min^–1, and a Hill coefficient of 1.8. In addition, WbpA has a K_0.5 for NAD⁺ of 220 µM, a k_cat of 86 min^–1, and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.

Received for publication, April 28, 2004 , and in revised form, June 16, 2004.

^* This research was supported by Grant MOP-14687 (to J. S. L.) from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work and should be considered co-first authors. Their names are listed in alphabetical order.

Recipient of a Doctoral Research Grant from the Canadian Institutes of Health Research.

^¶ Current address: The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.

^** Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology. To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 53823); Fax: 519-837-1802; E-mail: jlam{at}uoguelph.ca.

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