Understanding the Broad Substrate Repertoire of Nitroreductase Based on its Kinetic Mechanism [Molecular Biophysics]

April 4th, 2014 by Pitsawong, W., Hoben, J. P., Miller, A.-F.

The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in NR's broad substrate repertoire, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4°C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.9 ± 0.09 s-1, indicating one-step binding prior to the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s-1). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 727 ± 13 s-1 and a dissociation constant of 0.51 ± 0.04 mM. Thus we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain NR's broad substrate repertoire. Finally, we tested NR's ability to reduce para-hydroxylaminobenzoic acid (p-HABA), demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus the E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.