Advertisement

Biotechnology Letters

, Volume 41, Issue 10, pp 1155–1162 | Cite as

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation

  • Elsie M. Williams
  • Abigail V. Sharrock
  • Elizabeth L. Rylott
  • Neil C. Bruce
  • Joanna K. MacKichan
  • David F. AckerleyEmail author
Original Research Paper
  • 61 Downloads

Abstract

Objectives

To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants.

Results

To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis–Menten kinetic parameters with each dinitrotoluene substrate.

Conclusions

Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Keywords

Bioremediation Dinitrotoluene NADPH depletion assay NfsA NfsB Nitroreductase YfkO 

Notes

Acknowledgements

This work was supported by grants from the Royal Society of New Zealand Marsden Fund (VUW0704 and VUW1502), the UK Biotechnology and Biological Sciences Research Council (BB/P005713/1), and the Strategic Environmental Research and Development Program (ER-2723).

References

  1. Akiva E, Copp JN, Tokuriki N, Babbitt PC (2017) Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily. Proc Natl Acad Sci USA 114:E9549–E9558CrossRefGoogle Scholar
  2. Bolt HM, Degen GH, Dorn SB, Plottner S, Harth V (2006) Genotoxicity and potential carcinogenicity of 2,4,6-trinitrotoluene: structural and toxicological considerations. Rev Environ Heatl 21:217–228Google Scholar
  3. Copp JN, Williams EM, Rich MH, Patterson AV, Smaill JB, Ackerley DF (2014) Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs. Protein Eng Des Sel 27:399–403CrossRefGoogle Scholar
  4. Copp JN et al (2017) Engineering a multifunctional nitroreductase for improved activation of prodrugs and PET probes for cancer gene therapy. Cell Chem Biol 24:391–403CrossRefGoogle Scholar
  5. Dontsova KM, Pennington JC, Hayes C, Simunek J, Williford CW (2009) Dissolution and transport of 2,4-DNT and 2,6-DNT from M1 propellant in soil. Chemosphere 77:597–603CrossRefGoogle Scholar
  6. Hannink NK, Subramanian M, Rosser SJ, Basran A, Murray JA, Shanks JV, Bruce NC (2007) Enhanced transformation of TNT by tobacco plants expressing a bacterial nitroreductase. Int J Phytoremediation 9:385–401CrossRefGoogle Scholar
  7. Mayer KM, Arnold FH (2002) A colorimetric assay to quantify dehydrogenase activity in crude cell lysates. J Biomol Screen 7:135–140CrossRefGoogle Scholar
  8. Mowday AM et al (2016) Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy. Biochem Pharmacol 116:176–187CrossRefGoogle Scholar
  9. Prosser GA et al (2010) Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954. Biochem Pharmacol 79:678–687CrossRefGoogle Scholar
  10. Prosser GA et al (2013) Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A. Biochem Pharmacol 85:1091–1103CrossRefGoogle Scholar
  11. Rich MH, Sharrock AV, Hall KR, Ackerley DF, MacKichan JK (2018) Evaluation of NfsA-like nitroreductases from Neisseria meningitidis and Bartonella henselae for enzyme-prodrug therapy, targeted cellular ablation, and dinitrotoluene bioremediation. Biotechnol Lett 40:359–367CrossRefGoogle Scholar
  12. Roldan MD, Perez-Reinado E, Castillo F, Moreno-Vivian C (2008) Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol Rev 32:474–500CrossRefGoogle Scholar
  13. Van Dillewijn P, Couselo JL, Corredoira E, Delgado A, Wittich RM, Ballester A, Ramos JL (2008) Bioremediation of 2,4,6-trinitrotoluene by bacterial nitroreductase expressing transgenic aspen. Environ Sci Technol 42:7405–7410CrossRefGoogle Scholar
  14. Williams EM et al (2015) Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility. Biochem J 471:131–153CrossRefGoogle Scholar
  15. Zhang L, Rylott EL, Bruce NC, Strand SE (2019) Genetic modification of western wheatgrass (Pascopyrum smithii) for the phytoremediation of RDX and TNT. Planta 249:1007–1015CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.School of Biological SciencesVictoria University of WellingtonWellingtonNew Zealand
  2. 2.Department of ChemistryEmory UniversityAtlantaUSA
  3. 3.Centre for BiodiscoveryVictoria University of WellingtonWellingtonNew Zealand
  4. 4.Department of Biology, Centre for Novel Agricultural ProductsUniversity of YorkYorkUK

Personalised recommendations