Kim, Yong-Hak Yu, Myeong-Hee 2024-01-20T14:33:05Z 2024-01-20T14:33:05Z 2022-01-25 2012-06 1535-3893 https://pubs.kist.re.kr/handle/201004/129159 The effects of redox-sensitive proteins on Escherichia coli were investigated by overexpressing Pseudomonas 2-nitrobenzoate nitroreductase (NbaA) and its mutants. Overexpression of wild-type and mutant NbaA proteins significantly altered the sensitivity of E. coli to antibiotics and reactive oxygen species regardless of the enzyme activity for reduction of 2-nitrobenzoic acid. The overexpressed proteins rendered cells 100-10000-fold more sensitive to superoxide anion (O-2(center dot-))-generating paraquat and 10-100-fold more resistant to H2O2. A significant increase in intracellular levels of O-2(center dot-), but not H2O2, was observed during expression of wild-type and truncated (Delta 65-74, Delta 193-216, and Delta 65-74 Delta 193-216) NbaA. From two-dimensional nonreducing/reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry analyses, 29 abundant proteins in the cytoplasm were identified to form interchain disulfide bonds, when cells were exposed to polymyxin B. Of them, down-regulation and modifications of SodB, KatE, and KatG were strongly associated with elevated cellular O-2(center dot-) levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with, similar to 2-fold up-regulation of heterodimeric integration host factor, Ihf. Activity gel assays revealed significant reduction of glyceraldehyde-3-phosphate dehydrogenase with constant levels of 6-phosphogluconate dehydrogenase. These changes would support a high level of NADPH to reduce H2O2-induced disulfide bonds by forced expression of thioredoxin A via thioredoxin reductase. Thus, overexpression of wild-type and truncated NbaA partially compensates for the lack of KatE and KatG to degrade H2O2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O-2(center dot-) levels. English AMER CHEMICAL SOC Overexpression of Reactive Cysteine-Containing 2-Nitrobenzoate Nitroreductase (NbaA) and Its Mutants Alters the Sensitivity of Escherichia coli to Reactive Oxygen Species by Reprogramming a Regulatory Network of Disulfide-Bonded Proteins Article 10.1021/pr300221b 1 JOURNAL OF PROTEOME RESEARCH, v.11, no.6, pp.3219 - 3230 JOURNAL OF PROTEOME RESEARCH 11 6 3219 3230 N scie scopus 000304682500015 2-s2.0-84861793175 Biochemical Research Methods Biochemistry & Molecular Biology Article FLUORESCENS STRAIN KU-7 OXIDATIVE STRESS ACTIVE-SITE CELL-DEATH IN-VIVO SUPEROXIDE-DISMUTASE 2-COMPONENT SYSTEM RNA-POLYMERASE THIOREDOXIN CYTOPLASM disulfide bond reactive oxygen species sensitivity antimicrobial susceptibility superoxide anion hydrogen peroxide superoxide dismutase catalase-peroxidase regulatory pathway cell death signal