Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance.

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Nitroreductases (NRs) and ene-reductases (ERs) both utilize flavin mononucleotide cofactors but catalyze distinct reactions. NRs reduce nitroaromatics, whereas ERs reduce unsaturated C=C double bonds, and these functionalities are known to somewhat overlap. Recent studies on the ER xenobiotic reductase A (XenA) from Pseudomonas putida demonstrated the possibility of increasing NR activity with active site modifications. Structural comparison between NRs and ERs led us to hypothesize that active site cavity size plays an important role in determining enzyme functionality. Residues of ER KYE1 from Kluyveromyces lactis were selected to increase the binding pocket size, compensate for hydrogen bonding pattern changes, and eliminate ER activity. Single variants were screened, and promising mutations were combined. Variant F296A/Y275A showed a 100-fold improvement in NR specific activity over wild-type, and variant H191A/F296A/Y375A exhibited complete conversion to a NR.

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