SEARCH · Search PubMed

Results for “Nitroreductases”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed1996

Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.

We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1984

Studies on cis-trans isomerization of nitrofuran derivatives by bacterial nitroreductases.

cis-trans Isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)-acrylamide(AF-2) using Escherichia coli B/r and its two 5-nitro-2-furaldehyde semicarbazone (nitrofurazone)-resistant mutants was investigated. The isomerizing activity was detected in all three strains and markedly increased with acquiring resistance to nitro-furazone in intact cells and cell free extracts. Two distinct isomerases, nicotinamide adenine dinucleotide phosphate(NADPH)-dependent one with high activity and NAD(P)H-dependent with low activity were separated by Sephadex G-100 and Sepharose 4B columns. Both enzyme activities agreed with the nitrofurazone-reducing activity due to O2-sensitive nitroreductase as reported previously. Another nitroreductase, O2-insensitive one, was unable to isomerize cis AF-2 to trans form. These results suggest that bacterial cis-trans isomerases are not O2-insensitive nitroreductase, but O2-sensitive ones.

Bacteria↗

PubMed2002

Nitroreductase-based GDEPT.

Nitroreductases that metabolise aromatic nitro groups to hydroxylamines are attractive as enzymes for GDEPT because of the very large electronic change that this metabolism generates, providing an efficient switch that can be exploited to generate potent cytotoxins. While nitroreductase enzymes are widespread, nearly all the work using these in GDEPT has been with the nfsB gene product of Escherichia coli, an oxygen-insensitive flavin mononucleotide nitroreductase (NTR). Four classes of prodrugs for NTR have been described; dinitroaziridinylbenzamides, dinitrobenzamide mustards, 4-nitrobenzylcarbamates and nitroindolines. While some quinones are excellent substrates for NTR, none have been identified as potential GDEPT prodrugs. The most widely studied prodrug used for GDEPT in conjunction with NTR is the dinitroaziridinylbenzamide CB 1954. This shows high selectivity (>1000-fold) in cell lines transfected with NTR, has potent and long-lasting inhibition of NTR-transfected tumours in mice, and is in Phase I trial in conjunction with virally-delivered NTR enzyme. The related mustard SN 23862 has similar selectivity and superior bystander effects in animal models. Nitrobenzyl carbamates of a variety of cytotoxic amines (including aniline mustards, enediynes, duocarmycin analogues, pyrrolobenzodiazepines and the antitumour antibiotics doxorubicin, actinomycin D and mitomcyin C) are metabolised efficiently by NTR to the hydroxylamines, that fragment to release the amines. Nitroindoline derivatives of duocarmycins also show moderate selectivity for NTR-transfected cell lines in culture.

Animals↗

PubMed1984

Studies on nifurtimox nitroreductase activity in liver and other rat tissues.

Rat liver microsomes exhibit nifurtimox (NFX) nitroreductase activity, which is mostly NADPH-dependent and is completely abolished by heating and under an atmosphere of air. Pure carbon monoxide inhibits for 28% microsomal NFX nitroreductase activity while FAD 1 mM significantly enhances it. Smaller activities than in liver were found in brain, small intestine, testes, lung and heart. Rat liver cytosol also showed NFX nitroreductase activity using either hypoxanthine or N-methylnicotinamide as substrates. These activities were inhibited by allopurinol or menadione respectively. Results suggest that cytochrome P-450, NADPH cytochrome c reductase, xanthinoxidase and aldehyde oxidase are able to reduce NFX nitrogroups in rat liver and other tissues.

Animals↗

PubMed2005

Towards gene therapy in prosthesis loosening: efficient killing of interface cells by gene-directed enzyme prodrug therapy with nitroreductase and the prodrug CB1954.

BACKGROUND: Loosening is a major complication in prosthesis surgery. To stabilize loosened orthopedic implants, the interface tissue surrounding the implant must be removed. As an alternative to manual removal, we explored the possibility of removing the tissue by gene-directed enzyme prodrug therapy. In the current study we investigated whether interface cells can be transduced by an HAdV-5 vector carrying the E.coli-derived nitroreductase gene and sensitized to the prodrug CB1954. METHODS: The gene transfer efficiency into cultures of diploid human interface cells was tested by exposing these cells to various concentrations of Ad.CMV.LacZ. Subsequently, we studied the susceptibility of cells to the NTR/CB1954 combination. RESULTS: X-gal staining of the Ad.CMV.LacZ-transduced cell cultures revealed that, at 200 plaque-forming units (pfu)/cell, 74% of the cells expressed the LacZ gene. Infection with an NTR construct in interface cell lines resulted in a 60-fold sensitization to the prodrug CB1954. In addition we observed that iotrolan (Isovist) contrast medium had no effect on viability of the cells. However, the presence of the contrast medium completely inhibited adenovirus-mediated gene transfer. CONCLUSIONS: From these data we conclude that HAdV-5-based vectors carrying nitroreductase can be used to sensitize interface tissue. Instead of contrast medium the clinical protocol will use an alternative visualization procedure.

Adenoviridae↗

PubMed2001

Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase.

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) to 10(7) M(-1) s(-1). In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Aniline Compounds↗

PubMed1998

Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase.

Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4Cl as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing > 99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a novel visible region circular dichroism spectrum which has a small distinct negative band at 366 nm and a large positive ellipticity at 454 nm with a shoulder centered at 480 nm.

Base Sequence↗

PubMed2001

Purification and characterization of an enzyme from Mycobacterium sp. Pyr-1, with nitroreductase activity and an N-terminal sequence similar to lipoamide dehydrogenase.

Mycobacterium sp. Pyr-1 produces an enzyme with nitroreductase activity that reduces 1-nitropyrene and 4-nitrobenzoic acid to the corresponding aromatic amines. This enzyme was constitutive and required NADH; and its activity was enhanced by FAD. It was inhibited by antimycin A, dicumarol, and o-iodosobenzoic acid; and it was inactivated by ammonium sulfate precipitation. After purification to homogeneity, the protein produced a single band on native and SDS-polyacrylamide gels and had a single amino-terminal sequence. The N-terminal amino acid sequence was identical to the corresponding sequences of the lipoamide dehydrogenases of M. leprae, M. tuberculosis and Corynebacterium glutamicum. The amino-terminal sequence was also similar to lipoamide dehydrogenases from M. smegmatis and several other bacteria. The amino acid sequence of an internal peptide (12 of 13 amino acids) was nearly identical to the corresponding sequences of lipoamide dehydrogenases from M. leprae and M. tuberculosis and was similar to those of C. glutamicum, Streptomyces coelicolor and S. seoulensis. The data show that a unique lipoamide dehydrogenase in Mycobacterium sp. Pyr-1, which differs from classic (Type I) bacterial nitroreductases, reduces aromatic nitro compounds to aromatic amines.

Amino Acid Sequence↗

PubMed1987

Mutagenicity of halogenated and other substituted dinitrobenzenes in Salmonella typhimurium TA100 and derivatives deficient in glutathione (TA100/GSH-) and nitroreductase (TA100NR).

In a previous study, it was shown that 1-chloro-2,4-dinitrobenzene (CDNB) was less mutagenic in a glutathione (GSH)-deficient derivative of Salmonella typhimurium TA100 (TA100/GSH-) than in TA100 itself, suggesting that the mutagenicity of the compound is dependent on GSH, possibly mediated by the action of a bacterial nitroreductase(s) on the CDNB-GSH conjugate. In the present study a series of mutagenicity tests were performed to determine how CDNB could be activated after reaction with GSH. In liquid preincubation assays, strains TA100, TA100/GSH- and TA100NR, a nitroreductase-deficient derivative of TA100, were treated with CDNB and its fluoro and bromo analogues (FDNB and BDNB), further with its GSH conjugate (S-GSH-DNB) and possible metabolic products, such as S-cysteine-dinitrobenzene (S-Cys-DNB) and S-methyl-dinitrobenzene (S-methyl-DNB), and with 2 more analogues, O-methyl-dinitrobenzene (O-methyl-DNB) and dinitrobenzene (DNB). CDNB, FDNB and BDNB were found to be mutagenic in TA100 and TA100NR, while TA100/GSH- was much less sensitive to the mutagenic action of these halogenated dinitrobenzenes. DNB, O-methyl-DNB, S-methyl-DNB and S-Cys-DNB induced equal numbers of His+ revertants in TA100 and TA100/GSH-, but were not mutagenic in TA100NR. S-GSH-DNB showed no mutagenic activity in any of the 3 strains under the present experimental conditions. These results suggest that the halogenated aromatics may react with bacterial DNA and produce pre-mutagenic alterations according to 2 mechanisms: direct attack on the DNA through nucleophilic substitution (SN2) of the halogen atoms; activation through GSH conjugation and subsequent nitroreduction of the conjugate or its metabolic products to more reactive intermediates.

Bacterial Proteins↗

PubMed1989

A sensitive method for the detection of mutagenic nitroarenes: construction of nitroreductase-overproducing derivatives of Salmonella typhimurium strains TA98 and TA100.

'Classical nitroreductase' is an enzyme involved in the intracellular metabolic activation of mutagenic nitroarenes. The nitroreductase gene of Salmonella typhimurium TA1538 was cloned into pBR322 and the plasmids harboring the gene were introduced into TA98 and TA100. The resulting strains (YG1021 and YG1026) had more than 50 times higher nitrofurazone-reductase activity than TA1538 containing pBR322, and were extremely sensitive to the mutagenic action of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. These results indicate that the new strains permit the efficient detection of mutagenic nitroarenes.

Cloning, Molecular↗

PubMed1993

Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity.

Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium. The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S. typhimurium TA98 and TA100. The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains. These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.

Acetyltransferases↗

PubMed1991

Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities.

Salmonella typhimurium YG1021, YG1024, YG1026 and YG1029 are new derivatives of the Ames tester strains TA98 and TA100, with elevated 'classical' nitroreductase or acetyl-CoA:N-hydroxyarylamine O-acetyltransferase level. Thirty mutagens with different structures were tested using these strains and the sensitivities were compared with those of the conventional strains and of the enzyme-deficient strains. Elevated O-acetyltransferase activity of the indicator strains specifically increased their ability to detect the mutagenicity of aromatic nitro, amino and hydroxylamino compounds, whereas the strains with high nitroreductase activity were very sensitive to some nitroaromatics. The combined use of the isogenic tester strains with different metabolic capacities was quite useful to assess the intracellular metabolic activation and detoxification mechanisms of chemical mutagens.

Acetyltransferases↗

PubMed1986

Mutagenicity of the phenacetin metabolites: N-hydroxy-p-phenetidine and nitrosophenetol in S. typhimurium TA100 and derivatives deficient in nitroreductase or O-acetylase: probes for testing intrabacterial metabolic activation.

Two mutagenic metabolites of phenacetin, p-nitrosophenetol and N-hydroxy-p-phenetidine, were tested in S. typhimurium strains TA100, its nitroreductase-deficient derivative TA100NR, and O-acetylase-deficient strains TA100 Tn5-1,8-DNP1011 and -DNP1012 in the presence or absence of an exogenous metabolic activation system. The results indicate that bacterial nitroreductase(s) and O-acetylase(s), shown to be involved in the conversion of certain nitroarenes, are not required for the intrabacterial activation of the two phenacetin metabolites to bacterial mutagens. In view of the low reactivity of nitrosoarenes towards nucleophiles at neutrality, the mechanism by which they exert such a high mutagenic effect in S. typhimurium strains remains to be clarified, but is discussed.

Acetylesterase↗

PubMed2004

Nitroreductase catalyzed biotransformation of CL-20.

Previously, we reported that a salicylate 1-monooxygenase from Pseudomonas sp. ATCC 29352 biotransformed CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) (C(6)H(6)N(12)O(12)) and produced a key metabolite with mol. wt. 346 Da corresponding to an empirical formula of C(6)H(6)N(10)O(8) which spontaneously decomposed in aqueous medium to produce N(2)O, NH(4)(+), and HCOOH [Appl. Environ. Microbiol. (2004)]. In the present study, we found that nitroreductase from Escherichia coli catalyzed a one-electron transfer to CL-20 to form a radical anion (CL-20(-)) which upon initial N-denitration also produced metabolite C(6)H(6)N(10)O(8). The latter was tentatively identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4',5'-e]pyrazine [IUPAC] which decomposed spontaneously in water to produce glyoxal (OHCCHO) and formic acid (HCOOH). The rates of CL-20 biotransformation under anaerobic and aerobic conditions were 3.4+/-0.2 and 0.25+/-0.01 nmol min(-1)mg of protein(-1), respectively. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.8 nitrite ions, 3.3 molecules of nitrous oxide, 1.6 molecules of formic acid, 1.0 molecule of glyoxal, and 1.3 ammonium ions. Carbon and nitrogen products gave mass-balances of 60% and 81%, respectively. A comparative study between native-, deflavo-, and reconstituted-nitroreductase showed that FMN-site was possibly involved in the biotransformation of CL-20.

Aza Compounds↗

PubMed1997

Nitroreductase independent mutagenicity of 1-halogenated-2,4-dinitrobenzenes.

The 1-halogen substituted 2,4-dinitrobenzenes have been found to be mutagenic in Salmonella TA98 with an activity order of 1-fluoro > 1-chloro > 1-bromo > 1-iodo. This specific activity was not lowered in the nitroreductase deficient strain TA98NR and O-acetyltransferase deficient strain TA98/1,8-DNP6, indicating that the mutagenicity of these compounds is not dependent upon -NO2 reduction to -NHOH and its subsequent esterification. The activity was, rather, higher in the former and remained almost equal in the latter strain compared to TA98, suggesting these compounds to react directly with bacterial DNA. Further, the reaction of these halodinitrobenzenes with methionine, at physiological pH, resulting in the formation of 1-S-methyldinitrobenzene showed that these compounds have the ability to attack DNA directly through nucleophilic substitution of the halogen atom and the role of the bacterial nitroreductases for production of mutagenic lesions was not considered. It was, further, proposed that these compounds interact with DNA through SNAr mechanism instead of SN2 cited in literature.

Alkylating Agents↗

PubMed2000

Quantitative RT-PCR analysis of multiple genes encoding putative metronidazole nitroreductases from Helicobacter pylori.

Metronidazole (Mtz), a pro-drug, requires reductive activation by ferredoxin-like electron carrier proteins to kill bacteria and Mtz resistance is associated with a decrease or deficiency of Mtz nitroreductase activities in a target cell. Several genes encoding ferredoxin-like or -linked proteins such as pyruvate oxidoreductase (POR), ferredoxin oxidoreductase (FOR), ferredoxin (FdxA), ferredoxin-like protein (FdxB), flavodoxin (FldA) and oxygen insensitive nitroreductase (RdxA) have been identified from the complete genomic sequence of Helicobacter pylori. To understand the roles of these genes in H. pylori Mtz resistance, the gene expression for the proteins was examined using a method optimized for quantitative reverse transcription polymerase chain reaction (RT-PCR). The RT-PCR products of FOR and RdxA were significantly decreased in the total RNA prepared from H. pylori cultured in the presence of Mtz as compared to the total RNA prepared from H. pylori cultured without Mtz in the media. A slight decrease, however, in band intensity of the RT-PCR products of the POR and, to a lesser extent, FdxB was obtained in the presence of Mtz. In contrast, the RT-PCR products of the FdxA, FldA, and GalE (UDP-galactose 4-epimerase; a control gene) were unchanged in total RNA prepared from H. pylori cultured with or without Mtz in the culture media. These results suggest that Mtz resistance may also be acquired by decreasing the transcription of some genes involved in Mtz reductive activation, in addition to the mutation in some individual genes such as rdxA.

Base Sequence↗

PubMed2006

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase.

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.

Antineoplastic Agents↗