Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions.
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.
Hassett
D J
DJ
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA. Daniel.Hassett@UC.Edu
Ochsner
U A
UA
Groce
S L
SL
Parvatiyar
K
K
Ma
J F
JF
Lipscomb
J D
JD
D000332:Aerobiosis; D029681:Arabidopsis Proteins; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D017930:Genes, Reporter; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000378:metabolism
D029681:Arabidopsis Proteins; C081231:KATA protein, Arabidopsis; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; D001616:beta-Galactosidase; D016547:Kinesins