Understanding the Broad Substrate Repertoire of Nitroreductase Based on Its Kinetic Mechanism

作者：Pitsawong Warintra; Hoben John P; Miller Anne Frances^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2014, 289(22): 15203-15214.

DOI：10.1074/jbc.M113.547117

• Enzyme Kinetics
• Enzyme Mechanisms
• Flavin
• Flavoproteins
• Reductase
• Nitroreductase
• Substrate Specificity

Background: Nitroreductase reduces a broad range of nitroaromatics. Results: Steady-state and pre-steady-state kinetics were combined with tests for aminoaromatic product formation. Conclusion: Both half-reactions occur via a simple mechanism lacking detectable gating steps consistent with the broad substrate repertoire. Significance: Nitroreductase does not generate p-aminobenzoic acid and, therefore, appears not to reduce nitro groups to amines.
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 the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 degrees C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 +/- 0.09 s(-1), indicating one-step binding before 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 700 +/- 20 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 the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.

• 出版日期2014-5-30