A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation
- 61 Downloads
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 proteins enabled in vitro determination of Michaelis–Menten kinetic parameters with each dinitrotoluene substrate.
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.
KeywordsBioremediation Dinitrotoluene NADPH depletion assay NfsA NfsB Nitroreductase YfkO
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).
- 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