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PubMed1992

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase.

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

Antibodies↗
PubMed1992

A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

Cloning, Molecular↗
PubMed1990

Effect of lindane on nitroreductase and dechlorinase enzyme activity in the gastrointestinal tract.

A previously reported acceleration of parathion metabolism in the gastrointestinal (GI) tract of lindane-pretreated rats could have been due to either a prolonged residence time of parathion or increased GI nitroreductase activity or both. Thus to determine the effect on GI nitroreductase and dechlorinase activity, 20 mg/kg lindane or 535 mg/kg neomycin were administered daily, by gavage, to weanling F-344 rats. Enzyme activity in the small intestine and cecum were assayed after 2 weeks and 5 weeks of treatment. Neomycin treatment inhibited the activity of both enzymes in the cecum but had no significant effect on enzyme activity in the small intestine, suggesting the presence of mucosal nitroreductase and dechlorinase in the small intestine. In contrast, lindane, which had no effect on enzyme activity in the cecum, significantly increased nitroreductase activity in the small intestine after treatment for 5 weeks. This increased nitroreductase may account for the previously reported lindane-parathion interaction and could influence the metabolism, toxicity, and risk assessment of many other environmental nitro-compounds that become toxic, mutagenic or carcinogenic upon reduction of their nitro-groups.

Animals↗
PubMed2006

Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation.

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.

Animals↗
PubMed1999

Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli.

Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH. In this paper, nitroreductase A is induced in Escherichia coli by exposure to paraquat in a manner that depends on the expression of soxR. Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gene encoding nitroreductase A, but it was approximately 3-fold induced in the parental strain. Nitroreductase A thus appears to be a member of the soxRS regulon and probably contributes to the defenses against oxidative stress by minimizing the redox cycling attendant upon the univalent reduction of nitro compounds, quinones, and dyes.

Drug Resistance, Neoplasm↗
PubMed1991

Dinitropyrene nitroreductase activity of purified NAD(P)H-quinone oxidoreductase: role in rat liver cytosol and induction by Aroclor-1254 pretreatment.

Dinitropyrenes (DNP) are potent bacterial mutagens in the Ames test and genotoxins in cultured mammalian cells. Rat liver cytosol contains nitroreductases that are critical in the activation of DNP to the ultimate DNA-binding species. In order to study the nature and inducibility of liver cytosolic enzymes involved in the activation of DNP, cytosolic nitroreductase activities towards three DNP isomers (1,3-, 1.6- and 1,8-DNP) were determined in Aroclor-pretreated and untreated rats. Aroclor-1254 pretreatment resulted in up to 5-fold induction of cytosolic DNP nitroreductase. This induction was reflected in at least a 15-fold increase in cytosolic NAD(P)H-quinone oxidoreductase (NQOR) (E.C. 1.6.99.2) activity. The rates of nitroreduction for the three DNP isomers followed the order 1,6- greater than 1,8- greater than 1,3-DNP in all cases studied. 1,6-DNP nitroreductase coeluted with NQOR activity upon affinity purification. Highly purified NQOR catalyzed the NADH- and NADPH-dependent reduction of each of the three DNP isomers and displayed the same stereospecificity as the cytosolic activity. These results provide evidence that NQOR participates in the cytosolic nitroreduction of DNP and constitutes a major part of the total DNP nitroreductase activity upon induction of NQOR by Aroclor-1254 pretreatment. Thus, the role of NQOR in the metabolism of these mutagens depends significantly upon the degree to which this enzyme is induced.

Animals↗
PubMed1996

Investigation of alternative prodrugs for use with E. coli nitroreductase in 'suicide gene' approaches to cancer therapy.

The most commonly employed 'suicide' gene/prodrug system used in cancer gene therapy is the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir system. We have examined the efficacy of an alternative approach utilising the E. coli nitroreductase B enzyme with CB1954 and a variety of other prodrugs. V79 cells transfected with a nitroreductase expression vector were up to 770-fold more sensitive to CB1954 than control non-expressing cells. In general other prodrugs which were found by HPLC to act as substrates for purified E. coli nitroreductase also exhibited increased cytotoxicity against the nitroreductase-expressing cells, although this correlation was not absolute. In particular nitrofurazone (97-fold) and additional aromatic nitro-compounds (nine- to 50-fold) showed a large differential whereas the quinones and the antimetabolite, B-FU, were less effective (< three-fold). The results support the possibility of using nitroreductase and CB1954 for 'suicide gene' therapy and in addition suggest that alternative prodrugs, such as nitrofurazone, warrant further investigation in this novel approach.

Animals↗
PubMed1999

Nitroarene reduction and generation of free radicals by cell-free extracts of wild-type, and nitroreductase-deficient and -enriched Salmonella typhimurium strains used in the umu gene induction assay.

Studies of the enzymatic properties of cell-free extracts prepared from overnight cultures of the normal, and nitroreductase-deficient and -enriched strains of Salmonella typhimurium, designed for use in the umu gene induction assay of Oda et al. (1992), were undertaken in an effort to clarify the nature of nitroreductase deficiency in relation to mutagenicity. The ability of these strains to promote oxygen consumption and free radical intermediates of representative nitroarene substrates was measured, respectively, by oxygen polarography and electron spin resonance (ESR) spectroscopy. The substrates 4-nitropyridine N-oxide (4NPO) and 4-nitroquinoline N-oxide (4NQO) stimulated the rate and extent of NADH-dependent oxygen consumption catalyzed by cell-free extracts prepared from wild-type, and nitroreductase-deficient and -enriched strains. The extent of oxygen consumption was greater than stoichiometric with respect to the amount of nitroaromatic substrate, which implied one-electron reduction of 4NQO by these bacterial extracts and subsequent redox cycling with oxygen. ESR spectroscopy confirmed the production of free radical metabolites of the nitroarene substrates, which were inferred by the oxygen consumption studies. At equal protein concentrations the cell-free extracts of each strain catalyzed univalent reduction of 4NPO yielding the 59 line signal characteristic of the 4NPO nitro anion radical. This ESR signal was potently inhibited by the flavoprotein inhibitors CuSO4 and PCMB, albeit a twofold or higher concentration of both inhibitors was required to inhibit the signal produced by extract from the nitroreductase-deficient strain than that produced by the other strains. The results indicate that the nitroreductase-deficient strain of Salmonella typhimurium developed for use in the umu gene induction assay is not deficient in either one-electron nitro group or quinone reductase activity.

4-Nitroquinoline-1-oxide↗
PubMed1994

Mutagenicity testing in Salmonella typhimurium strains possessing both the His reversion and Ara forward mutation systems and different levels of classical nitroreductase or O-acetyltransferase activities.

The induction of forward mutations to L-arabinose resistance (AraR) and of reversions to histidine prototrophy (His+) can be quantitatively compared in Salmonella typhimurium BA strains. The BA bacteria carry the araD531 allele required for the Ara assay and a his auxotrophy (hisD3052 or hisG46) required for the His assay. In this study, 2 new sets of BA indicator strains have been constructed in order to combine the Ara forward and the His reverse mutation assays of S. typhimurium with deficiency, or over-production, in either classical nitroreductase or O-acetyltransferase for mutagenicity testing of nitro-containing chemicals. Nine mutagens with different chemical structures were tested to compare the specific mutagenic sensitivities of the new constructions with those of the parental and of the conventional TA indicator bacteria. The Ara test, which responded with high sensitivity to all chemicals tested, revealed important differences between the standard tester strains TA98 and TA100 with respect to the activation of mutagens considered to be dependent on classical nitroreductase activity. Total correspondence was found between the specific mutagenic sensitivities of the defective and the overproducing bacteria in the genetic background of TA98 but not in that of TA100. In the genetic background of TA100, chemicals such as nitrofurantoin and nitrofurazone displayed 10-fold reduced mutagenicity to the "classical nitroreductase" defective strain without increasing mutagenicity to the corresponding overproducing bacteria. This discrepancy might be attributed to the greater nitroreduction capability of strain TA100 (68.12 nmole/min/mg protein) as compared to TA98 (24.42 nmole/min/mg protein), by assuming that nitrofurantoin and nitrofurazone are such good substrates for classical nitroreductase that the additional enzyme activity produced from the corresponding overexpressing plasmid when present in TA100 no longer affected their metabolic activation. We propose that the Ara forward mutation test carried out in the set of over-producing bacteria constructed in the genetic background of TA98 might play a role for routine testing of large number of samples. The isogenic defective strains could be used in cases of uncertain results with the corresponding over-producing bacteria. Finally, the reversion of the his alleles accompanying the Ara assay in the BA strains could play a role in assessing the presence of mixtures of chemicals with different mutagenic specificity in samples of environmental relevance such as urban air, foods, and water.

Arabinose↗
PubMed1995

Role of classical nitroreductase and O-acetyltransferase on the mutagenicity of nifurtimox and eight derivatives in Salmonella typhimurium.

This study investigates the mutagenicity of nifurtimox (NFX) and eight analogues in Salmonella typhimurium indicator strains that possess different levels of classical nitroreductase or O-acetyltransferase activities. The NFX analogues tested replace the 3-methyl-4-yl-tetrahydro-1,4-thiazine-1,1-dioxide group of the parent compound with the following other groups: indazol-1-yl (1G); pyrazol-1-yl (1B); benzimidazol-1-yl (1E); 1,2,4-triazol-4-yl (1D); 1-methyl-3-methylthio-1,2,4-triazol-4-yl-5-thione (1I); 3,5-bis(methylthio)-1,2,4-triazol-4-yl (1H); 1-adamantyl (ADA); and 4,6-diphenylpyridin-1-yl-2-one (1K). In the genetic backgrounds of the standard Ames tester strains TA98 and TA100, these bacteria combine the L-arabinose resistance forward mutation assay (Ara test) with a deficiency or overproduction of either nitroreduction or O-acetylation. The Ara test revealed, in agreement with previous findings, important differences between TA98 and TA100 and demonstrated, moreover, that these genetic differences are of significance in mutagenicity testing with nitrofuran compounds. The Ara test also indicated dissimilarities between the metabolic activation of NFX and its analogues, these compounds being classified in three different groups according to their mutagenicity toward strain BA14 (genetic background of TA98) and its derivatives. The first group included analogues (1G, 1E, 1I, and ADA) that showed similar mutagenic potency in all bacterial strains. These compounds are considered not to be substrates for both classical nitroreductase and O-acetyltransferase. The second group included compounds (analogues 1B and 1K, and the reference drug NFX) with increased mutagenicity toward the strain overproducing the classical nitroreductase, and/or reduced mutagenicity toward the corresponding deficient bacteria. These compounds are considered to be activated by the classical nitroreductase. The third group (analogues 1D and 1H) was activated by bacterial O-acetyltransferase, and consequently showed increased and decreased mutagenicity with the particular overproducer or deficient bacterial strain, as compared to their isogenic parentals. Previous reports have pointed out interest in NFX analogue 1H as a promising candidate for the replacement of NFX. The present study further enhances the putative interest of compound 1H, based on the different metabolic activation pathway exhibited by this analogue as compared to the parental drug, NFX.

Acetyltransferases↗
PubMed1995

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

Aniline Mustard↗
PubMed1991

Role of bacterial nitroreductase and O-acetyltransferase in urine mutagenicity assay of rats exposed to 2,4,6-trinitrotoluene (TNT).

The urine mutagenicity of rats exposed to 2,4,6-trinitrotoluene (TNT) by i.p. injection was studied in the Salmonella assay using indicator strains with various levels of 'classical' nitroreductase or acetyl-CoA:N-hydroxylarylamine O-acetyltransferase activity. The strains used were the conventional Salmonella typhimurium TA98, nitroreductase-deficient TA98NR and -overproducing YG1021, and O-acetyltransferase-deficient TA98/1,8-DNP6 and -overproducing YG1024. TA98, YG1021 and YG1024 clearly detected the increase of direct urine mutagenicity. A slight increase of mutagenicity was also detected with metabolic activation in YG1021 and YG1024. High levels of both nitroreductase and O-acetyltransferase significantly increased the sensitivity of the indicator strain to the mutagenicity of urine caused by TNT exposure, while the nitroreductase- or O-acetyltransferase-deficient strains gave negative responses.

Acetyltransferases↗
PubMed2001

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Bacteria↗
PubMed2001

Fluorogenic substrates for the detection of microbial nitroreductases.

AIMS: To synthesize and evaluate fluorogenic substrates for the detection of microbial nitroreductases. These substrates, all based on 7-nitrocoumarin, may be reduced to form fluorescent aminocoumarins. METHODS AND RESULTS: Thirty pathogenic microbial strains, including both bacteria and yeasts, were examined for nitroreductase activity in a whole-cell assay. All strains readily reduced each of the seven substrates to generate fluorescence, suggesting the widespread presence of nitroreductase activity in pathogenic bacteria. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: These novel substrates facilitate the direct detection of nitroreductase activity and have potential as sensitive indicators of microbial growth.

Bacteria↗
PubMed1982

Evidence for the existence of distinct nitroreductases in Salmonella typhimurium: roles in mutagenesis.

Previous studies on the mutagenicity of nitroheterocyclics and nitrated polycyclic aromatic hydrocarbons had indicted the probable existence in Salmonella typhimurium of a multiplicity of nitroreductase activities of varying specificities which are required for the expression of the mutagenicity of nitro-containing chemicals. In the present study evidence is presented that these activities reside in different gene products: (a) strains totally lacking in the nitroreductase which recognizes niridazole and related substances are not mutagenized by this group of chemicals and yet they are fully responsive to the mutagenic action of dinitropyrenes; (b) double mutants which have lost both types of specificities can be constructed. Finally, the presence of a third type of nitroreductase with a specificity for 4-nitroquinoline-1-oxide is implied by the finding that the nitroreductase-deficient strains described herein retain full sensitivity to the mutagenic action of this chemical.

Mutagens↗
PubMed1982

Mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxy-phenacetin and their respective deacetylated metabolites in nitroreductase deficient Salmonella TA98FR and TA100FR.

The mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin and their respective deacetylated metabolites, N-hydroxy-2-aminofluorene and 2-nitrosofluorene, and N-hydroxyphenetidine and rho-nitrosophenetole was determined in nitroreductase deficient Salmonella tester strains TA 98FR and TA100FR. The mutagenicity of N-hydroxy-2-acetylaminofluorene medicated by either rat liver microsomes or rat liver 105 000 g supernatant fractions was no different in either TA98 (nitroreductase proficient) or TA98FR (nitroreductase deficient). Similarly the mutagenicity of N-hydroxyphenacetin mediated by hamster liver microsomes was not affected by either the presence or absence of nitroreductase activity in TA100. N-Hydroxy-2-aminofluorene and 2-nitrosofluorene were equipotent direct acting mutagens in both TA98 and TA98FR, as were both N-hydroxyphenetidine and rho-nitrosophenetole in TA100 and TA 100FR. Ascorbate (5 mM) and NADPH (1 mM) had no significant effect on the mutagenicity of either N-hydroxy-2-acetylaminofluorene, N-hydroxy-2-aminofluorene, or 2-nitrosofluorene in TA98 or TA98FR whereas ascorbate and NADPH markedly inhibited the mutagenicity of both N-hydroxyphenetidine and rho-nitrosophenetole in both TA100 and TA100FR. Ascorbate appears to be inhibiting the mutagenicity of N-hydroxyphenetidine and rho-nitrosophenetole as a result of the nonenzymatic chemical reduction of these compounds to non-mutagenic derivatives.

2-Acetylaminofluorene↗
PubMed1985

Nitroreductase-dependent mutagenicity of p-nitrophenylhydroxylamine and its N-acetyl and N-formyl hydroxamic acids.

p-Nitrophenylhydroxylamine (NPH) and two hydroxamic acids derived from it were synthesized and subjected to mutagenicity testing in Salmonella typhimurium strains TA98, TA98NR, TA1538 and TA1538NR. In addition, p-dinitrobenzene (DNB), p-nitroaniline (NA) and p-nitroacetanilide (AcNA) were simultaneously examined for mutagenic action against these four tester strains. NPH, its N-acetyl (AcNPH) and N-formyl (FoNPH) derivatives, and also DNB displayed strong mutagenic action to the nitroreductase-containing strains, TA98 and TA1538. NPH was the most potent chemical in this series against both of these strains, while the two hydroxamic acids AcNPH and FoNPH, and also DNB displayed approximately the same degree of mutagenicity. In the nitroreductase-deficient strains, TA98NR and TA1538NR, the mutagenicity of these four compounds was markedly reduced. The necessity for nitroreduction in order to activate these promutagens is fairly certain; however, the lack of mutagenicity of NA and AcNA towards all four tester strains made the interpretation of these data somewhat more complicated. Several possible bioactivation pathways were presented, with one mechanism in particular being proposed. This mechanism requires only that the strong electron-withdrawing nitro group be converted to an electron-donating group by bacterial nitroreductase. Such a mechanism is unique for the bioactivation of nitro aromatics by nitroreductase, since the enzymatic reduction need not produce the intermediary hydroxylamine metabolite.

Acetylation↗
PubMed2003

Vibrio harveyi nitroreductase is also a chromate reductase.

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5alpha and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5alpha NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5alpha NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K(m) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 micro M, respectively. The V(max) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V(max)/K(m) value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30 degrees C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).

Cloning, Molecular↗