Understanding the broad substrate repertoire of nitroreductase based on its kinetic mechanism.
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 °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.
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055.
Warintra
W
Pitsawong
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055.
John P
JP
Hoben
From the Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055 afm@uky.edu.
Anne-Frances
AF
Miller
D000332:Aerobiosis; D000588:Amines / Q000378:metabolism; D000693:Anaerobiosis; D001673:Biodegradation, Environmental; D004138:Dinitrocresols / Q000378:metabolism; D016972:Enterobacter cloacae / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009579:Nitrobenzoates / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D000588:Amines; D004138:Dinitrocresols; D029968:Escherichia coli Proteins; D009579:Nitrobenzoates; D009243:NAD; C024132:4,6-dinitro-o-cresol; C116054:NfsB protein, E coli; D009601:Nitroreductases; C008629:4-nitrobenzoic acid
Enzyme Kinetics; Enzyme Mechanisms; Flavin; Flavoproteins; Nitroreductase; Reductase; Substrate Specificity