TY - JOUR
T1 - Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase
T2 - A cautionary note on use of xylE reporter fusions under aerobic conditions
AU - Hassett, D. J.
AU - Ochsner, U. A.
AU - Groce, S. L.
AU - Parvatiyar, K.
AU - Ma, J. F.
AU - Lipscomb, J. D.
PY - 2000
Y1 - 2000
N2 - Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H2O2) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding β-galactosidase) or -xylE fusion plasmids to H2O2 stimulated β-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 H2O2, while a 10,000-fold excess was required for similar inhibition of β-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H2O2 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.
AB - Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H2O2) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding β-galactosidase) or -xylE fusion plasmids to H2O2 stimulated β-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 H2O2, while a 10,000-fold excess was required for similar inhibition of β-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H2O2 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.
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U2 - 10.1128/AEM.66.9.4119-4123.2000
DO - 10.1128/AEM.66.9.4119-4123.2000
M3 - Article
C2 - 10966438
AN - SCOPUS:0033823729
SN - 0099-2240
VL - 66
SP - 4119
EP - 4123
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
IS - 9
ER -