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

Biotechnology Letters
Elsie M WilliamsDavid F Ackerley

Abstract

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. 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...Continue Reading

References

May 15, 2002·Journal of Biomolecular Screening·Kimberly M Mayer, Frances H Arnold
Feb 6, 2008·International Journal of Phytoremediation·Nerissa K HanninkNeil C Bruce
Mar 22, 2008·FEMS Microbiology Reviews·María Dolores RoldánConrado Moreno-Vivián
Oct 23, 2008·Environmental Science & Technology·Pieter Van DillewijnJuan L Ramos
Sep 5, 2009·Chemosphere·Katerina M DontsovaClint W Williford
Jul 6, 2014·Protein Engineering, Design & Selection : PEDS·J N CoppD F Ackerley
Oct 4, 2015·The Biochemical Journal·Elsie M WilliamsDavid F Ackerley
Jul 28, 2016·Biochemical Pharmacology·Alexandra M MowdayAdam V Patterson
Oct 29, 2017·Proceedings of the National Academy of Sciences of the United States of America·Eyal AkivaPatricia C Babbitt

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Datasets Mentioned

BETA
AAM73721

Methods Mentioned

BETA
transgenic

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GraphPad Prism
GraphPad

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