Bacterial oxygen-insensitive NAD(P)H:nitroaromatic oxidoreductase (NR; EC 1.6.99.7) from Enterobacter cloacae is a homodimeric enzyme in which each of the two symmetry-related active sites occurs in the interface between monomers and contains a flavin mononucleotide (FMN). NR catalyzes two-electron reduction of nitroaromatic compounds to corresponding nitroso- and, subsequently, hydroxylamino products. NR has an unusually broad substrate specificity range, which may be related to protein dynamics and/or a simple transient kinetic mechanism.To test the possible role of mechanism in NR's broad substrate specificity, the reduction kinetics of NR by NAD(P)H at pH7.50 were investigated using stopped-flow techniques. The observed linear dependence on NADH and NADPH concentration indicates a one-step reduction mechanism with bimolecular rate constants of 8.95 � 105 M-1s-1 and 2.38 � 105 M-1s-1, respectively (or that only the linear portion of a hyperbolic dependence was detected). There is no evidence for a distinct binding step in which specificity might be enforced. The oxidative half-reaction of NR with p-nitrobenzoic acid (PNBA) showed a hyperbolic dependence on the PNBA concentration, indicating a simple one-step binding prior to the flavin oxidation step (1.46 � 0.02 s-1). The simple transient kinetics data in both the reductive and oxidative half-reactions can thus provide a possible explanation for the broad substrate range of NR. There is no direct evidence for hydride transfer in this enzyme. Moreover the X-ray crystal structure of oxidized NR shows PNBA bound with the nitro group more than 5 � from the flavin N(5) which is the presumed source of hydride. Therefore, primary and solvent kinetic isotope effects are being investigated to elucidate the hydride transfer mechanism.

Transient kinetics of nitroreductase from Enterobacter cloacae and kinetic isotope effects as a tool for probing hydride transfer

246th National Meeting (2013)

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