Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase : A cautionary note on use of xylE reporter fusions under aerobic conditions

• Published in: Applied and environmental microbiology 2000-09, Vol.66 (9), p.4119-4123
• Main Authors: HASSETT, Daniel J, OCHSNER, Urs A, GROCE, Stephanie L, PARVATIYAR, Kislay, MA, Ju-Fang, LIPSCOMB, John D
• Format: Article
• Language: English
• Subjects: Aerobiosis; Arabidopsis Proteins; beta-Galactosidase - antagonists & inhibitors; beta-Galactosidase - metabolism; Biological and medical sciences; Biotechnology; Catechol 2,3-Dioxygenase; Dioxygenases; Enzymology and Protein Engineering; Fundamental and applied biological sciences. Psychology; Genes; Genes, Reporter; Genetic engineering; Genetic technics; Hydrogen; Hydrogen Peroxide - pharmacology; katA gene; katB gene; Kinesin - genetics; Kinesin - metabolism; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Oxygen; Oxygenases - antagonists & inhibitors; Oxygenases - genetics; Oxygenases - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Pseudomonas putida; Pseudomonas putida - enzymology; Pseudomonas putida - genetics; Pseudomonas putida - growth & development; Recombinant Fusion Proteins - drug effects; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Synthetic digonucleotides and genes. Sequencing; XylE gene
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.66.9.4119-4123.2000

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.