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

The Journal of Biological Chemistry
Wayne L MillerJoseph S Lam

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 oligomerizati...Continue Reading

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Citations

Jun 21, 2013·Molecular Microbiology·Robert G Lees-MillerMario F Feldman
Oct 21, 2005·Protein Expression and Purification·Patrycja K NiemiecShayan Sharif
Jul 19, 2008·Analytical Biochemistry·Seema C Namboori, David E Graham
Feb 24, 2015·Biotechnology Advances·Frederik De BruynMarjan De Mey
Aug 28, 2009·Innate Immunity·Jerry D KingJoseph S Lam
Jul 31, 2019·Antimicrobial Agents and Chemotherapy·Qingye XuXiaoting Hua
Dec 11, 2007·The Journal of Biological Chemistry·Wayne L MillerJoseph S Lam
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Feb 27, 2018·PLoS Pathogens·Paul A BeareRobert A Heinzen
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