Biochemical Characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas Aeruginosa PAO1

J Biol Chem. 2004 Sep 3;279(36):37551-8. doi: 10.1074/jbc.M404749200. Epub 2004 Jun 28.

Abstract

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 NH(4)(+) 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 microM, 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 microM, 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Biopolymers
  • Carbohydrate Dehydrogenases / isolation & purification
  • Carbohydrate Dehydrogenases / metabolism*
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • DNA Primers
  • Electrophoresis, Capillary
  • Kinetics
  • Microscopy, Electron
  • Nuclear Magnetic Resonance, Biomolecular
  • O Antigens / biosynthesis*
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / immunology*

Substances

  • Biopolymers
  • DNA Primers
  • O Antigens
  • Carbohydrate Dehydrogenases
  • UDP-N-acetylglucosamine dehydrogenase