Nitrobenzene reductase

Nitrobenzene reductase activity has been detected in the fat body, gut, and Malpighian tubules of the Madagascar cockroach, G. portentosa (Rose and Young, 1973). Anaerobic conditions are essential for activity. The enzymes in the microsomes are strongly NADH dependent, whereas those in the soluble fraction are strongly NADPH dependent. Activity is enhanced by the addition of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) or riboflavin. It appears that the true substrate for the nitroreductase is FMN and that the reduction of the nitro compounds occurs nonenzymatically (Figure 8.15). Similar results are obtained using azofuchsin as substrate. 

Reductive biotransformations of several compounds such as polyhalogenated, keto, nitro and azo derivatives, are catalysed by a variety of enzymes which differ according to the substrates and the species. The liver cytochrome P-450-dependent drug metabolizing system is capable of reducing Af-oxide, nitro and azo bonds, whereas the cytosolic nitrobenzene reductase activity is mainly due to cytochrome P-450 reductase, which transforms nitrobenzene into its hydroxylamino derivative. NADPH cytochrome c reductase is also able to catalyse the reduction of nitro compounds. These metabolic conversions may also be brought about by gastrointestinal anaerobic bacteria. 

Protein B from M. capsulatus (Bath) not only increases the product yields, but also influences the rate constant for the single turnover reaction of Hred with nitrobenzene (51,67). The pseudo-first-order rate constant increases up to 33-fold when Hred is titrated with protein B. Neither addition of reductase to Hox or Hred, nor addition of protein B and reductase to Hred, could similarly affect the rate constant. These... 

The reduction of nitro groups may also be catalyzed by microsomal reductases and gut bacterial enzymes. The reduction passes through several stages to yield the fully reduced primary amine, as illustrated for nitrobenzene (Fig. 4.39). The intermediates are nitrosobenzene and phenylhydroxylamine, which are also reduced in the microsomal system. These intermediates, which may also be produced by the oxidation of aromatic amines (Fig. 4.21), are involved in the toxicity of nitrobenzene to red blood cells after oral administration to rats. The importance of the gut bacterial reductases in this process is illustrated by the drastic reduction in nitrobenzene toxicity in animals devoid of gut bacteria, or when nitrobenzene is given by the intraperitoneal route. 

A condition described as "hereditary methemoglobinemia" may result from a genetic defect (Goldstein et al. 1969). The enzyme methemoglobin reductase is absent and persons are hypersensitive to any substances such as nitrite or aniline derivatives capable of producing methemoglobinemia. The trait is inherited as an autosomal recessive allele. Thus either sex may exhibit the trait which is ordinarily detected by the presence of cyanosis at birth. Such individuals would be extremely sensitive to the effects of nitrobenzene. 

Most studies in the microbial metabolism of nitroaromatic compounds used aerobic microorganisms. In most cases no mineralization of nitroaromatics occurs, and only superficial modifications of the structures are reported. However, under anaerobic sulfate-reducing conditions, the nitroaromatic compounds reportedly undergo a series of reductions with the formation of amino compounds. For example, trinitrotoluene under sulfate-reducing conditions is reduced to triaminotoluene by the enzyme nitrite reductase, which is commonly found in many Desulfovibrio spp. The removal of ammonia from triaminotoluene is achieved by reductive deamination catalyzed by the enzyme reductive deaminase, with the production of ammonia and toluene. Some sulfate reducers can metabolize toluene to (X) sub 2. Similar metabolic processes could be applied to other nitroaromatic compounds like nitrobenzene, nitrobenzoic acids, nitrophenols, and aniline. Many methanogenic bacteria can reduce nitroaromatic compounds to amino compounds. 

In addition to the oxidative systems, liver microsomes also contain enzyme systems that catalyze the reduction of azo and nitro compounds to primary amines. A number of azo compounds, such as Prontosil ahd sulfasalazine (Fig. 10.16), are converted to aromatic primary amines by azoreductase, an NADPFI-dependent enzyme system in the liver microsomes. Evidence exists for the participation of CYP450 in some reductions. Nitro compounds (e.g., chloramphenicol and nitrobenzene) are reduced to aromatic primary amines by a nitroreductase, presumably through hitrosamine and hydroxylamine intermediates. These reductases are not solely responsible for the reduction of azo ahd hitro compouhds reductioh by the bacterial flora ih the anaerobic environment of the intestine also may occur. 

Nitro reducing activity with TNT as the substrate has been observed in some aerobic microorganisms. A nitroaryl reductase able to reduce several nitrobenzenes, 3,5-dinitroben-zoic acid, and TNT has been purified from Neurospora crassa (52). Enzymes in crude... 

Bryant and DeLuca (5) purified and characterized a Type I nitroreductase from Enterobacter cloacae. The protein is a monomer of approximately 27 kDa, it has a loosely bound FMN cofactor and uses NAD(P)H as an electron donor. The substrate range of the enzyme includes nitrofurans, nitrobenzenes, nitrotoluenes and quinones. The enzyme appears to produce the hydroxylamino derivative of nitroflirazone under aerobic conditions, but the product was not thoroughly characterized. Under anaerobic conditions the product is the amine. The authors did not test the effect of metals on enzyme activity, and no inhibition studies were reported. Therefore, it is not known if this enzyme is a metalloprotein. The gene encoding the Enterobacter reductase was cloned and sequenced (7). The authors found a 651 base pair open reading frame corresponding to a protein of 23.9 kDa. Comparison of the Enterobacter and Salmonella amino acid sequences revealed 88% sequence identity between the two proteins (7). 

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