Oxygen-insensitive Nitroreductase Research Articles

Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine

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.

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine).

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.

Review article: nitroimidazole resistance in Helicobacter pylori

The efficacy of a nitroimidazole-containing regimen for the treatment of Helicobacter pylori infection is decreased by nitroimidazole resistance. Nitroimidazoles are meta- bolized by H. pylori by several nitro-reductases of which an oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene is the most important. Null mutations in this gene are associated with resistance. Susceptibility testing to nitroimidazoles may give variable results. This is not only related to the slow growth under specific conditions, but also to variability in the activity of the other nitroreductases and the ability to deactivate toxic metabolites of an NI and to repair DNA damage. Moreover, co-infections with resistant and susceptible bacteria are frequently found. The presence of nitroimidazole resistance is related to the previous use of this drug. The prevalence of resistance is rising and nowadays 10-50% of the isolates are resistant. Resistance reduces the efficacy of a treatment regimen to a variable degree. This is related to efficacy of the other components of the regimen and the treatment duration. Whether a nitroimidazole is still effective in resistant strains remains unresolved. When nitroimidazole resistance is present, a nitro-imidazole-containing regimen should be avoided or a regimen with other highly effective components should be used.

Oxygen-insensitive nitroreductases of E. coli do not reduce protein nitrotyrosine

Oxygen-insensitive nitroreductases of E. coli do not reduce protein nitrotyrosine

Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli.

Nitroheterocyclic and nitroaromatic compounds constitute an enormous range of chemicals whose potent biological activity has significant human health and environmental implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the nfsA and nfsB genes of a large number of nitrofuran-resistant mutants of Escherichia coli and have correlated mutation with cell extract nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an nfsA mutation, while the increased resistance associated with second-step mutants is a consequence of an nfsB mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, especially with regard to the identification of flavin mononucleotide binding sites. We show that expression of plasmid-carried nfsA and nfsB genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and 49%, respectively, contained insertion sequence elements in nfsA and nfsB. IS1 integrated in both genes, while IS30 and IS186 were found only in nfsA and IS2 and IS5 were observed only in nfsB. Insertion hot spots for IS30 and IS186 are indicated in nfsA, and a hot spot for IS5 insertion is evident in nfsB. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in nfsA and nfsB.

Conversion of NfsA, the major Escherichia coli nitroreductase, to a flavin reductase with an activity similar to that of Frp, a flavin reductase in Vibrio harveyi, by a single amino acid substitution.

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

Gene cloning, purification, and characterization of NfsB, a minor oxygen-insensitive nitroreductase from Escherichia coli, similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri.

nfsB, encoding a minor oxygen-insensitive nitroreductase, was isolated by PCR using primers corresponding to two amino acid sequences conserved among the major flavin reductase from Vibrio fischeri and classical nitroreductases from Salmonella typhimurium and Enterobacter cloacae. The gene product, NfsB, was purified to homogeneity from extracts of Escherichia coli cells overexpressing it. NfsB was found to be situated at 13 min on the E. coli map. Biochemical analysis indicated NfsB to be a polypeptide having a calculated molecular weight of 23,904, capable of forming a homodimer and associated tightly with FMN as a prosthetic group. Although it exhibited a lower affinity to the NfsB apoenzyme than FMN, FAD could serve as an effective substitute for FMN. It was also shown that NfsB has a broad electron acceptor specificity and is associated with a low level of the NAD(P)H-flavin oxidoreductase. The NfsB catalysis obeys the ping pong Bi-Bi mechanism. The Km value for NADH varied depending on the second substrate used.

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.

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.