Abstract
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.
Highlights
The reductive productsof several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic
The purified nitroreductase can utilizeeither NADHorNADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes aswellas quinones
As a result a plethora of information concerning the toxic, mutagenic, and carcinogenic properties of a large number of nitroaromatic compounds has accumulated over the past few decades
Summary
To date studies concerning the characterizationof these enzymes have been performed using partially purified preparations of the enzymes. This is in part due to therelatively minute quantities present within the cells. Additional proof of the homogeneity of the final preparation was indicated by presence of a single peak during chromatofocusing of the purified enzyme (data notshown). Analysis of the Q300 anion-exchange elution profile indicated the presence of at least three distinct peaks of nitroreductase activity. Multiple peaks of nitroreductase activity have been noted when cell free extracts of E. coli or S. typhimurium were fractionated by DEAE-cellulose chroma-
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