title: Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine.
abstract: The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds. We tested the hypothesis that these nitroreductases are capable of reducing 3-nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli. E. coli homogenates were incubated with nitrated proteins and the level of 3-nitrotyrosine immunoreactivity was assayed by Western blotting. Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-nitrotyrosine in protein, peptide, or free amino acid. Stimulation of nfsA nfsB activity with paraquat had no effect on 3-nitrotyrosine reduction. Nonlethal exposure of E. coli to peroxynitrite/CO(2) resulted in the reproducible nitration of tyrosine residues in endogenous proteins. The degree of 3-nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains. These results indicate that the nfsA and nfsB enzymes do not reduce 3-nitrotyrosine.
authors.affiliation: Stokes Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Lightfoot
authors.forename: D
authors.initials: D
authors.lastname: Shuman
authors.forename: H
authors.initials: H
authors.lastname: Ischiropoulos
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology*; D002458:Cell Fractionation; D019008:Drug Resistance, Neoplasm; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D029968:Escherichia coli Proteins; D009566:Nitrates / Q000378:metabolism; D009601:Nitroreductases / Q000096:biosynthesis / Q000502:physiology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000633:toxicity*; D010269:Paraquat / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009566:Nitrates; D010455:Peptides; D019163:Reducing Agents; C002744:3-nitrotyrosine; D014443:Tyrosine; C029492:aromatic NADH-dependent nitroreductase; C116053:NfsA protein, E coli; C116054:NfsB protein, E coli; D009601:Nitroreductases; D010269:Paraquat; D010100:Oxygen