A Comprehensive Characterization of the Nitroreductase Superfamily

The Nitroreductase (NTR) superfamily has been studied for over 50 years, offering a wide variety of biotechnological applications, including cancer prodrug therapy, biocatalysis, and bioremediation(Ackerley et al., 2004; Jaberipour et al., 2010). However, functional studies on the NTR superfamily have primarily focused on two E. coli enzymes, NfsA and NfsB, and their homologs, leading to an incomplete and misleading classification system (Roldan et al., 2008). Seeking to address this issue, we are conducting a thorough and comprehensive characterization of the Nitroreductase superfamily. By analyzing >600 enzymes and >25 different substrates, we are conducting the first investigations into the massive, intriguing, and uncharacterized sequence-space of this superfamily.

To gain insight into the overall organization of the superfamily, we make use of Sequence Similarity Networks(SSN), which enable us to examine relationships within massive and diverse sets of sequences for which more traditional methods, like phylogenetic trees, would be prohibitive. Using SSNs, the NTR superfamily can be divided into 23 distinct subgroups based on their protein-sequence similarity. By selecting and screening representative enzymes from each of the subgroups in aerobic and anaerobic conditions, we have been able to functionally annotate various segments of the newly discovered sequence-space.

References:

1. Ackerley, D.F., Gonzalez, C.F., Keyhan, M., Blake II, R., and Matin, A. (2004). Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction. Environmental microbiology, 6(8), 851-860.
2. Jaberipour, M., Vass. S.O., Guise, C.P., Grove, J.I., Knox, R.J., Hu.L, Hyde. El, and Searle. P.F. (2010). Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: effects of combining beneficial single mutations. Biochem. Pharmacol, 79(2), 102-111.
3. Roldan, M.D., Perez-Reinaldo, E., Castillo, F., and Moreno-Vivian, C. (2008). Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol. Rev., 32(3), 474-500.