Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.
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.
Miller
Wayne L
WL
Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Wenzel
Cory Q
CQ
Daniels
Craig
C
Larocque
Suzon
S
Brisson
Jean-Robert
JR
Lam
Joseph S
JS
D001483:Base Sequence; D001704:Biopolymers; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D007700:Kinetics; D008854:Microscopy, Electron; D019906:Nuclear Magnetic Resonance, Biomolecular; D019081:O Antigens / Q000096:biosynthesis*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000276:immunology*
D001704:Biopolymers; D017931:DNA Primers; D019081:O Antigens; D002237:Carbohydrate Dehydrogenases; C046670:UDP-N-acetylglucosamine dehydrogenase