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Khaled A S Alrasheid - One of the best experts on this subject based on the ideXlab platform.
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comparative evaluation of genotoxicity induced by Nitrofurazone in two ciliated protozoa by detecting dna strand breaks and dna protein crosslinks
Ecological Indicators, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Liang Zhou, Xudong Cui, Khaled A S AlrasheidAbstract:Abstract Although organism-specific factors related to individual indicator organisms have hampered the use of bioassays for the evaluation of environmental risk in practice, the importance of understanding organism-specific factors when selecting model organisms has also not yet been fully recognized. In this work, genotoxicity was evaluated in the ciliated protozoa, Euplotes vannus and Pseudokeronopsis rubra, when exposed to graded doses of Nitrofurazone for several discrete durations. Genotoxicity was expressed based on the LD50 and was determined by assessing DNA strand breaks (through alkaline comet assay) and DNA–protein crosslinks (DPCs), by means of a KCl–SDS precipitation assay. It was found that E. vannus generally had lower LD50's than P. rubra (P 0.05). The relationship between these parameters was reversed in the case of P. rubra. Biphasic dose–response relationships were generally detected between Nitrofurazone and genotoxicity parameters, however, parameters for DNA strand breaks presented significantly positive correlations between each other (P
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recognizing the importance of exposure dose response dynamics for ecotoxicity assessment Nitrofurazone induced antioxidase activity and mrna expression in model protozoan euplotes vannus
Environmental Science and Pollution Research, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Shuxing Liu, Khaled A S AlrasheidAbstract:The equivocality of dose–response relationships has, in practice, hampered the application of biomarkers as a means to evaluate environmental risk, yet this important issue has not yet been fully recognized or explored. This paper evaluates the potential of antioxidant enzymes in the ciliated protozoan Euplotes vannus for use as biomarkers. Dose–response dynamics, together with both the enzyme activity and the gene expression of the antioxidant enzymes, superoxide dismutase, and glutathione peroxidase, were investigated when E. vannus were exposed to graded doses of Nitrofurazone for several discrete durations. Mathematical models were explored to characterize the dose–response profiles and, specifically, to identify any equivocality in terms of endpoint. Significant differences were found in both enzyme activity and messenger RNA (mRNA) expression in the E. vannus treated with Nitrofurazone, and the interactions between exposure dosage and duration were significant. Correlations between enzyme activity, mRNA expression, and Nitrofurazone dose varied with exposure duration. Particularly, the dose–responses showed different dynamics depending on either endpoint or exposure duration. Our findings suggest that both the enzyme activity and the gene expression of the tested antioxidant enzymes can be used as biomarkers for ecotoxicological assessment on the premise of ascertaining appropriate dosage scope, exposure duration, endpoint, etc., which can be achieved by using dose–response dynamics.
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characterizing dose responses of catalase to Nitrofurazone exposure in model ciliated protozoan euplotes vannus for ecotoxicity assessment enzyme activity and mrna expression
Ecotoxicology and Environmental Safety, 2014Co-Authors: Liang Zhou, Xiaofeng Lin, Khaled A S AlrasheidAbstract:In environmental studies, some biological responses, known as biomarkers, have been used as a powerful bioassay tool for more than four decades. Disparity between enzyme activity and mRNA abundance leads to correlation equivocality, which makes the application of biomarkers for environmental risk assessment more complicated. This study investigates this disparity in the case of catalase when used as a biomarker for detecting ecotoxicity induced by antibiotics in aquatic ecosystems. In particular, dose–responses for catalase activity and mRNA expression abundance were investigated in Euplotes vannus which were exposed to graded doses of Nitrofurazone for several discrete durations, and dose–response models were developed to characterize the dose–response dynamics. Significant differences were found in both catalase activity and mRNA expression abundance among the E. vannus treated with Nitrofurazone. Catalase activity showed a hormetic-like effect in terms of dose–response, characterized by a biphasic relationship which was more clearly evident after a longer exposure period, while mRNA expression abundance increased linearly with the exposure duration. Additionally, the correlation between catalase activity and mRNA expression abundance reversed along with the duration of exposure to Nitrofurazone. Taken together, our results demonstrate that catalase mRNA expression offers a more straightforward dose–response model than enzyme activity. Our findings suggest that both catalase enzyme activity and mRNA expression abundance can be used jointly as bioassay tools for detecting ecotoxicity induced by Nitrofurazone in aquatic ecosystems.
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use of rapd to detect dna damage induced by Nitrofurazone in marine ciliate euplotes vannus protozoa ciliophora
Aquatic Toxicology, 2011Co-Authors: Liang Zhou, Xiaofeng Lin, Khaled A S AlrasheidAbstract:Abstract The random amplified polymorphic DNA (RAPD) assay was evaluated as a potential tool to detect the ecotoxicity induced by Nitrofurazone in marine ciliate, Euplotes vannus. The data revealed a reduction in viability of the test ciliates with increasing Nitrofurazone concentration in the range of 0–24 mg l−1 and time of exposure from 24 to 96 h. The Nitrofurazone treated ciliates were subjected to DNA damage analysis by RAPD assay. Among the 33 test RAPD primers used in this study, 11 primers with 60–70% GC content produced unique polymorphic band patterns. A total of 213 bands of 155–3317 bp in molecular size range were observed in the untreated cells. In comparison with the control ciliates, the Nitrofurazone treated groups showed differences in RAPD profiles with respect to the band intensity, disappearance of bands and appearance of new bands of amplified DNA. The variation of RAPD profiles showed both the time- and concentration-dependent relationships. The data suggested significant genomic template instability, which corresponds well with the viability of the test ciliates. Thus the results demonstrated the potential of the RAPD assay for application as a powerful tool for detecting genotoxicity induced by fishy drugs in aquatic environment.
Eva I Hyde - One of the best experts on this subject based on the ideXlab platform.
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kinetic and structural characterisation of escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate cb1954
Journal of Molecular Biology, 2007Co-Authors: Paul R Race, Andrew L Lovering, Scott A White, Peter F Searle, Jane I Grove, Christopher W Wrighton, Eva I HydeAbstract:Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic Nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over Nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over Nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.
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structural and mechanistic studies of escherichia coli nitroreductase with the antibiotic Nitrofurazone reversed binding orientations in different redox states of the enzyme
Journal of Biological Chemistry, 2005Co-Authors: Paul R Race, Andrew L Lovering, Richard M Green, Abdelmijd Ossor, Scott A White, Peter F Searle, Christopher J Wrighton, Eva I HydeAbstract:The antibiotics Nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for Nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-Nitrofurazone complex, Nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound Nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.
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structural and mechanistic studies of escherichia coli nitroreductase with the antibiotic Nitrofurazone reversed binding orientations in different redox states of the enzyme
Journal of Biological Chemistry, 2005Co-Authors: Paul R Race, Andrew L Lovering, Richard M Green, Abdelmijd Ossor, Scott A White, Peter F Searle, Christopher J Wrighton, Eva I HydeAbstract:The antibiotics Nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ∼10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for Nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-Nitrofurazone complex, Nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound Nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.
Xiaofeng Lin - One of the best experts on this subject based on the ideXlab platform.
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comparative evaluation on combined ecotoxic effects of Nitrofurazone and nh 4 ci using two ciliated protists as model organisms population dynamics and growth rates
Journal of Ocean University of China, 2021Co-Authors: Shuxing Liu, Alan Warren, Xin Chen, Xiaofeng LinAbstract:Ecotoxic effects of antibiotics or ammonium have been confirmed independently in aquatic animals, but few studies have investigated their combined effects. In aquaculture ecosystems, these pollutants frequently coexist, and often in high concentrations. In this study, the combined effects of antibiotic Nitrofurazone and NH4Cl on the population dynamics and growth rates of two species of ciliated protists, Euplotes vannus and Pseudokeronopsis rubra, were investigated. Profiles of the dose-responses were visualized, and interactions between the two pollutants were quantified by the response surface method (RSM). Results showed that 1) the dynamics of the population growth differed significantly between the testd ciliates and varied with the concentrations of the pollutants; 2) the relative growth rate (RGR) of both ciliates decreased significantly with increased pollutant concentrations, while the difference in RGR between the two ciliates was not significant; 3) RSM analysis demonstrated an additive effect of Nitrofurazone and NH4Cl on the RGR in both ciliates. In brief, ecotoxic effects can be caused by Nitrofurazone and ammonium independently on the two test ciliates, and such effects can be strengthened when they present at the same time. These findings offer a valuable reference for evaluating combined ecotoxic effects caused by multiple pollutants in aquaculture ecosystems.
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evaluation of biomarkers for ecotoxicity assessment by dose response dynamic models effects of Nitrofurazone on antioxidant enzymes in the model ciliated protozoan euplotes vannus
Ecotoxicology and Environmental Safety, 2017Co-Authors: Yazhen Hong, Yalin Tan, Yang Meng, Hao Yang, Yu Zhang, Alan Warren, Xiaofeng LinAbstract:Understanding dose-responses is crucial for determining the utility of biomarkers in ecotoxicity assessment. Nitrofurazone is a broad-spectrum antibiotic that is widely used in the aquaculture industry in China despite its detrimental effects on ecosystems. Potential dose-response models were examined for the effect of Nitrofurazone on two antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), in the ciliated protozoan Euplotes vannus. This was achieved by measuring enzyme activity and gene expression profiling of SOD and GPx in ciliate cells exposed to Nitrofurazone at doses ranging from 0 to 180mgl-1 for 6h, 12h, 18h and 24h. Dose-response dynamics were characterized by mathematical models. Results showed that: 1) dose-response patterns differed significantly among the tested endpoints, Nitrofurazone concentrations and durations of exposure; 2) GPx activity was the best candidate biomarker because of its linear dose-response relationship; 3) SOD activity and mRNA relative expression levels of GPx and SOD are also candidate biomarkers but their dose-responses were non-linear and therefore more difficult to interpret; 4) partitioning the dose-response dynamic model by piecewise function can help to clarify the relationships between biological endpoints. This study demonstrates the utility of dynamic model analysis and the potential of antioxidant enzymes, in particular GPx activity, as a candidate biomarkers for environmental monitoring and risk assessment of Nitrofurazone in the aquaculture industry.
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comparative evaluation of genotoxicity induced by Nitrofurazone in two ciliated protozoa by detecting dna strand breaks and dna protein crosslinks
Ecological Indicators, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Liang Zhou, Xudong Cui, Khaled A S AlrasheidAbstract:Abstract Although organism-specific factors related to individual indicator organisms have hampered the use of bioassays for the evaluation of environmental risk in practice, the importance of understanding organism-specific factors when selecting model organisms has also not yet been fully recognized. In this work, genotoxicity was evaluated in the ciliated protozoa, Euplotes vannus and Pseudokeronopsis rubra, when exposed to graded doses of Nitrofurazone for several discrete durations. Genotoxicity was expressed based on the LD50 and was determined by assessing DNA strand breaks (through alkaline comet assay) and DNA–protein crosslinks (DPCs), by means of a KCl–SDS precipitation assay. It was found that E. vannus generally had lower LD50's than P. rubra (P 0.05). The relationship between these parameters was reversed in the case of P. rubra. Biphasic dose–response relationships were generally detected between Nitrofurazone and genotoxicity parameters, however, parameters for DNA strand breaks presented significantly positive correlations between each other (P
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recognizing the importance of exposure dose response dynamics for ecotoxicity assessment Nitrofurazone induced antioxidase activity and mrna expression in model protozoan euplotes vannus
Environmental Science and Pollution Research, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Shuxing Liu, Khaled A S AlrasheidAbstract:The equivocality of dose–response relationships has, in practice, hampered the application of biomarkers as a means to evaluate environmental risk, yet this important issue has not yet been fully recognized or explored. This paper evaluates the potential of antioxidant enzymes in the ciliated protozoan Euplotes vannus for use as biomarkers. Dose–response dynamics, together with both the enzyme activity and the gene expression of the antioxidant enzymes, superoxide dismutase, and glutathione peroxidase, were investigated when E. vannus were exposed to graded doses of Nitrofurazone for several discrete durations. Mathematical models were explored to characterize the dose–response profiles and, specifically, to identify any equivocality in terms of endpoint. Significant differences were found in both enzyme activity and messenger RNA (mRNA) expression in the E. vannus treated with Nitrofurazone, and the interactions between exposure dosage and duration were significant. Correlations between enzyme activity, mRNA expression, and Nitrofurazone dose varied with exposure duration. Particularly, the dose–responses showed different dynamics depending on either endpoint or exposure duration. Our findings suggest that both the enzyme activity and the gene expression of the tested antioxidant enzymes can be used as biomarkers for ecotoxicological assessment on the premise of ascertaining appropriate dosage scope, exposure duration, endpoint, etc., which can be achieved by using dose–response dynamics.
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characterizing dose responses of catalase to Nitrofurazone exposure in model ciliated protozoan euplotes vannus for ecotoxicity assessment enzyme activity and mrna expression
Ecotoxicology and Environmental Safety, 2014Co-Authors: Liang Zhou, Xiaofeng Lin, Khaled A S AlrasheidAbstract:In environmental studies, some biological responses, known as biomarkers, have been used as a powerful bioassay tool for more than four decades. Disparity between enzyme activity and mRNA abundance leads to correlation equivocality, which makes the application of biomarkers for environmental risk assessment more complicated. This study investigates this disparity in the case of catalase when used as a biomarker for detecting ecotoxicity induced by antibiotics in aquatic ecosystems. In particular, dose–responses for catalase activity and mRNA expression abundance were investigated in Euplotes vannus which were exposed to graded doses of Nitrofurazone for several discrete durations, and dose–response models were developed to characterize the dose–response dynamics. Significant differences were found in both catalase activity and mRNA expression abundance among the E. vannus treated with Nitrofurazone. Catalase activity showed a hormetic-like effect in terms of dose–response, characterized by a biphasic relationship which was more clearly evident after a longer exposure period, while mRNA expression abundance increased linearly with the exposure duration. Additionally, the correlation between catalase activity and mRNA expression abundance reversed along with the duration of exposure to Nitrofurazone. Taken together, our results demonstrate that catalase mRNA expression offers a more straightforward dose–response model than enzyme activity. Our findings suggest that both catalase enzyme activity and mRNA expression abundance can be used jointly as bioassay tools for detecting ecotoxicity induced by Nitrofurazone in aquatic ecosystems.
Yazhen Hong - One of the best experts on this subject based on the ideXlab platform.
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evaluation of biomarkers for ecotoxicity assessment by dose response dynamic models effects of Nitrofurazone on antioxidant enzymes in the model ciliated protozoan euplotes vannus
Ecotoxicology and Environmental Safety, 2017Co-Authors: Yazhen Hong, Yalin Tan, Yang Meng, Hao Yang, Yu Zhang, Alan Warren, Xiaofeng LinAbstract:Understanding dose-responses is crucial for determining the utility of biomarkers in ecotoxicity assessment. Nitrofurazone is a broad-spectrum antibiotic that is widely used in the aquaculture industry in China despite its detrimental effects on ecosystems. Potential dose-response models were examined for the effect of Nitrofurazone on two antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), in the ciliated protozoan Euplotes vannus. This was achieved by measuring enzyme activity and gene expression profiling of SOD and GPx in ciliate cells exposed to Nitrofurazone at doses ranging from 0 to 180mgl-1 for 6h, 12h, 18h and 24h. Dose-response dynamics were characterized by mathematical models. Results showed that: 1) dose-response patterns differed significantly among the tested endpoints, Nitrofurazone concentrations and durations of exposure; 2) GPx activity was the best candidate biomarker because of its linear dose-response relationship; 3) SOD activity and mRNA relative expression levels of GPx and SOD are also candidate biomarkers but their dose-responses were non-linear and therefore more difficult to interpret; 4) partitioning the dose-response dynamic model by piecewise function can help to clarify the relationships between biological endpoints. This study demonstrates the utility of dynamic model analysis and the potential of antioxidant enzymes, in particular GPx activity, as a candidate biomarkers for environmental monitoring and risk assessment of Nitrofurazone in the aquaculture industry.
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comparative evaluation of genotoxicity induced by Nitrofurazone in two ciliated protozoa by detecting dna strand breaks and dna protein crosslinks
Ecological Indicators, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Liang Zhou, Xudong Cui, Khaled A S AlrasheidAbstract:Abstract Although organism-specific factors related to individual indicator organisms have hampered the use of bioassays for the evaluation of environmental risk in practice, the importance of understanding organism-specific factors when selecting model organisms has also not yet been fully recognized. In this work, genotoxicity was evaluated in the ciliated protozoa, Euplotes vannus and Pseudokeronopsis rubra, when exposed to graded doses of Nitrofurazone for several discrete durations. Genotoxicity was expressed based on the LD50 and was determined by assessing DNA strand breaks (through alkaline comet assay) and DNA–protein crosslinks (DPCs), by means of a KCl–SDS precipitation assay. It was found that E. vannus generally had lower LD50's than P. rubra (P 0.05). The relationship between these parameters was reversed in the case of P. rubra. Biphasic dose–response relationships were generally detected between Nitrofurazone and genotoxicity parameters, however, parameters for DNA strand breaks presented significantly positive correlations between each other (P
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recognizing the importance of exposure dose response dynamics for ecotoxicity assessment Nitrofurazone induced antioxidase activity and mrna expression in model protozoan euplotes vannus
Environmental Science and Pollution Research, 2015Co-Authors: Yazhen Hong, Xiaofeng Lin, Shuxing Liu, Khaled A S AlrasheidAbstract:The equivocality of dose–response relationships has, in practice, hampered the application of biomarkers as a means to evaluate environmental risk, yet this important issue has not yet been fully recognized or explored. This paper evaluates the potential of antioxidant enzymes in the ciliated protozoan Euplotes vannus for use as biomarkers. Dose–response dynamics, together with both the enzyme activity and the gene expression of the antioxidant enzymes, superoxide dismutase, and glutathione peroxidase, were investigated when E. vannus were exposed to graded doses of Nitrofurazone for several discrete durations. Mathematical models were explored to characterize the dose–response profiles and, specifically, to identify any equivocality in terms of endpoint. Significant differences were found in both enzyme activity and messenger RNA (mRNA) expression in the E. vannus treated with Nitrofurazone, and the interactions between exposure dosage and duration were significant. Correlations between enzyme activity, mRNA expression, and Nitrofurazone dose varied with exposure duration. Particularly, the dose–responses showed different dynamics depending on either endpoint or exposure duration. Our findings suggest that both the enzyme activity and the gene expression of the tested antioxidant enzymes can be used as biomarkers for ecotoxicological assessment on the premise of ascertaining appropriate dosage scope, exposure duration, endpoint, etc., which can be achieved by using dose–response dynamics.
Paul R Race - One of the best experts on this subject based on the ideXlab platform.
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kinetic and structural characterisation of escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate cb1954
Journal of Molecular Biology, 2007Co-Authors: Paul R Race, Andrew L Lovering, Scott A White, Peter F Searle, Jane I Grove, Christopher W Wrighton, Eva I HydeAbstract:Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic Nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over Nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over Nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.
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structural and mechanistic studies of escherichia coli nitroreductase with the antibiotic Nitrofurazone reversed binding orientations in different redox states of the enzyme
Journal of Biological Chemistry, 2005Co-Authors: Paul R Race, Andrew L Lovering, Richard M Green, Abdelmijd Ossor, Scott A White, Peter F Searle, Christopher J Wrighton, Eva I HydeAbstract:The antibiotics Nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for Nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-Nitrofurazone complex, Nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound Nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.
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structural and mechanistic studies of escherichia coli nitroreductase with the antibiotic Nitrofurazone reversed binding orientations in different redox states of the enzyme
Journal of Biological Chemistry, 2005Co-Authors: Paul R Race, Andrew L Lovering, Richard M Green, Abdelmijd Ossor, Scott A White, Peter F Searle, Christopher J Wrighton, Eva I HydeAbstract:The antibiotics Nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant ∼10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for Nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-Nitrofurazone complex, Nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound Nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.