Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1

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DOIResolve DOI: http://doi.org/10.1074/jbc.M404749200
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TypeArticle
Journal titleJournal Of Biological Chemistry
Volume279
Issue36
Pages3755137558; # of pages: 8
Subjectacid; analysis; anions; antigen; binding; biosynthesis; capillary; chromatography; electron; electrophoresis; encodes; filtration; gene; HPLC; kinetic; microbiology; microscopy; NMR spectroscopy; nucleotides; O antigen; protein; Pseudomonas aeruginosa; serotype; SPECTROSCOPY; sugar; tertiary structure; ultracentrifugation
AbstractWbpA (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 date
LanguageEnglish
AffiliationNRC Institute for Biological Sciences; National Research Council Canada
Peer reviewedNo
NRC numberMILLER2004
NPARC number9367266
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Record identifierd16e2dec-9d37-4752-9027-c67b305e6ca8
Record created2009-07-10
Record modified2016-05-09
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