Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions.
Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions.
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儿茶酚-2,3-双加氧酶的过氧化氢敏感性：在有氧条件下使用 xylE 报告融合体的注意事项。
DOI：
10.1128/aem.66.9.4119-4123.2000
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发表时间：
2000
影响因子：
4.4
通讯作者：
Lipscomb,JD
中科院分区：
文献类型：
--
作者：
Hassett,DJ;Ochsner,UA;Groce,SL;Parvatiyar,K;Ma,JF;Lipscomb,JD
恶臭假单胞菌的儿茶酚-2,3-双加氧酶 (C23O) 由 thexylE 基因编码，当用作基因融合构建体中的报告基因时，被发现对过氧化氢 (H2O2) 敏感。将含有katA-orkatB-lacZ（编码β-半乳糖苷酶）或-xylE融合质粒的铜绿假单胞菌katAorkatA katB突变体暴露于H2O2刺激了β-半乳糖苷酶活性，而在这些菌株中几乎没有或没有可检测到的C23O活性。暴露于等摩尔 H2O2 5 分钟后，超过 95% 的 C23O 活性消失，而类似的 β-半乳糖苷酶抑制需要 10,000 倍过量。 C23O 亚硝酰配合物的电子顺磁共振谱表明，H2O2 几乎按化学计量氧化了必需的活性位点亚铁离子，从而导致活性丧失。我们的结果表明，在有氧条件下解释来自 xylEreporter 融合的数据时要小心，特别是在存在氧化应激或使用过氧化氢酶缺陷菌株时。
Catechol-2,3-dioxygenase (C23O) ofPseudomonas putida, encoded by thexylEgene, was found to be sensitive to hydrogen peroxide (H2O2) when used as a reporter in gene fusion constructs. Exposure ofPseudomonas aeruginosa katAorkatA katBmutants harboringkatA- orkatB-lacZ(encoding β-galactosidase) or -xylEfusion plasmids to H2O2stimulated 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 H2O2nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived fromxylEreporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.