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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J.T.P. was supported by grant No.IIP-0969003 from the National Science Foundation to the I/UCRC Center for Pharmaceutical Development (CPD). K.L.F. gratefully acknowledges support via a President’s Undergraduate Research Award (PURA). W.P. was supported by an RCTF fellowship from the University of Kentucky. A.S.B. and A.F.M. acknowledge support from NSF I/UCRC grants IIP-1540017 and IIP-1063879, respectively, to the Center for Pharmaceutical Development.
Supplementary Materials: The following are available online, four tables providing (1,2) lists of compounds studied, (3) surface areas buried in the NR dimers and (4) list of all NR structures and sequences used. Nine figures providing (1) sequence similarity network, (2) calibration and validation of E°c calculations, (3) dependence of amine formation on electron withdrawal (E°c), substrate volume or log(P), (4) dependence of rate constants on substituent constants, (5) sequential identification of NR subgroups on the basis of distinguishing structure, (6) numbering of the positions of the flavin right, and that of NADH, (7) comparisons of the shapes and sizes of the different subgroups’ active sites, (8) distribution of amino acid conservation in the structure, and (9) a multiple sequence alignment of the NRs whose structures were compared as well as supporting sequences.
Miller, Anne-Frances; Park, Jonathan T.; Ferguson, Kyle L.; Pitsawong, Warintra; and Bommarius, Andreas S., "Informing Efforts to Develop Nitroreductase for Amine Production" (2018). Chemistry Faculty Publications. 125.