Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

Bai, Jing; Yang, Jun; Liu, Peiyu; Yang, Qing

doi:10.1016/j.procbio.2015.01.029

Cited by 18 publications

(13 citation statements)

References 51 publications

(59 reference statements)

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“…Further research is necessary to substantiate this hypothetical pathway. Several studies (7,26) demonstrated that the E. coli nitroreductases NfsA and NfsB exhibit a significantly greater reduction rate of the nitro derivatives on the para position than on the ortho position of TNT. Assuming a similar reduction mechanism for DNT, this could explain the accumulation of 4A2NT rather than 2A4NT.…”

Section: Discussionmentioning

confidence: 99%

Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives

Shemer

Yagur‐Kroll

Hazan

et al. 2018

Appl Environ Microbiol

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DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an bioreporter strain engineered to detect traces of DNT and have demonstrated that the gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in , have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces is 2,4,5-trihydroxytoluene. We further show that cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells. The information provided in this article sheds new light both on the microbial biodegradability of nitroaromatic compounds and on the metabolic capabilities of By doing so, it also clarifies the pathway leading to the previously unexplained induction of the gene by 2,4-dinitrotoluene, an impurity that accompanies 2,4,6-trinitrotoluene (TNT)-based explosives. Our improved understanding of these processes will serve to molecularly enhance the performance of a previously described microbial bioreporter of buried landmines and other explosive devices, in which the gene promoter serves as the sensing element.

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Section: Discussionmentioning

confidence: 99%

Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives

Shemer

Yagur‐Kroll

Hazan

et al. 2018

Appl Environ Microbiol

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“…When Phe124 was mutated to a small residue (Ser or Ala), the FMN reduction activity of NfsB was increased significantly (to about 118‐fold that of the wild‐type enzyme, and even threefold that of FRase I) . When Phe123 was mutated to Ala, the catalytic activity toward all tested substrates was markedly improved . It is concluded that the removal of, or single amino acid substitutions, at positions 123 and 124 remove steric constraints preventing large flavins, such as FMN and FAD, from entering the substrate pocket, but cause no substantial change in the basic structure of the active site.…”

Section: Resultsmentioning

confidence: 98%

“…The crystal structures of NfsB (PDB code 1DS7, 1ICR) showed that these two helices constituted the entrance to the substrate pocket and influenced the substrate specificity and activity of NfsB . In particular, the importance of two phenylalanines (Phe123 and Phe124) located in helix α6 has been demonstrated using site‐directed mutagenesis . Wild‐type NfsB possesses little FMN reduction activity, whereas its homolog FRase I from V. fischeri had a considerable level of FMN reduction activity.…”

Section: Resultsmentioning

confidence: 99%

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001

Yang

Bai

et al. 2018

Biotech and App Biochem

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A nitroreductase-encoded gene from an efficient nitro-reducing bacterium Streptomyces mirabilis DUT001, named snr, was cloned and heterogeneously expressed in Escherichia coli. The purified Streptomyces nitroreductase SNR was a homodimer with an apparent subunit molecular weight of 24 kDa and preferred NADH to NADPH as a cofactor. By enzyme incubation and isothermal calorimetry experiments, flavin mononucleotide (FMN) was found to be the preferred flavin cofactor; the binding process was exothermic and primarily enthalpy driven. The enzyme can reduce multiple nitro compounds and flavins, including antibacterial drug nitrofurazone, priority pollutants 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates 3-nitrophthalimide, 4-nitrophthalimide, and 4-nitro-1,8-naphthalic anhydride. Among the substrates tested, the highest activity of k /K (0.234 μM Sec ) was observed for the reduction of FMN. Multiple sequence alignment revealed that the high FMN reduction activity of SNR may be due to the absence of a helix, constituting the entrance to the substrate pocket in other nitroreductases.

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“…However, such systems provide limited control over the reaction outcome, and in several cases the enzyme responsible for amine formation has been shown to be other than a NR [ 60 , 61 , 62 , 63 ]. Moreover, the extent to which the amine accumulates depends on the ambient reduction potential [ 7 , 40 , 64 ]. Nevertheless, a route to formation remains a prerequisite for any accumulation.…”

Section: Resultsmentioning

confidence: 99%

“…Numerous efforts are underway to develop nitroreductase enzymes to activate prodrugs [ 1 , 2 ], remediate pollutants [ 3 , 4 , 5 , 6 , 7 ] and generate building-blocks for high-value pharmaceuticals [ 8 ]. However, reduction of nitrated aromatics has been a challenge to chemists since the original work by Haber in 1898 [ 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

Informing Efforts to Develop Nitroreductase for Amine Production

et al. 2018

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Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance.

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Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis

Cited by 18 publications

References 51 publications

Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives

Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001

Informing Efforts to Develop Nitroreductase for Amine Production

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