Evaluation of NfsA-like nitroreductases from Neisseria meningitidis and Bartonella henselae for enzyme-prodrug therapy, targeted cellular ablation, and dinitrotoluene bioremediation
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To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene.
NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His6-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O2-insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate.
NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.
KeywordsCB1954 Dinitrotoluene Gene-directed enzyme-prodrug therapy Metronidazole NfsA Nitroreductase PnbA
This work was partially supported by grants from the Lottery Health Research Fund and the Wellington Medical Research Fund (2014/234) to JKM, and the Royal Society of New Zealand Marsden Fund (VUW1502 to DFA). MHR, KRH and AVS were supported by Victoria University of Wellington PhD Scholarships, with MHR additionally supported by the Cancer Society of New Zealand and AVS by a Dick and Mary Earle Scholarship in Technology.
Supplementary Table 1—IC50 evaluation for E. coli strains expressing wild type nitroreductases.
Supplementary Fig. 1—Kinetics of purified nitroreductases with CB1954.
Supplementary Fig. 2—Kinetics of purified nitroreductases with metronidazole.
Supplementary Fig. 3—Kinetics of purified nitroreductases with 2,4-dinitrotoluene.
Supplementary Fig. 4—Kinetics of purified nitroreductases with 2,6-dinitrotoluene.