Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae.
The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.
Bryant
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C
Department of Chemistry, University of California, San Diego, La Jolla 92093.
DeLuca
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D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004754:Enterobacter / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009601:Nitroreductases / Q000235:genetics / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; C029492:aromatic NADH-dependent nitroreductase; D009601:Nitroreductases