title: Reduction of quinones and nitroaromatic compounds by Escherichia coli nitroreductase A (NfsA): Characterization of kinetics and substrate specificity.
abstract: NfsA, a major FMN-associated nitroreductase of E. coli, reduces nitroaromatic compounds via consecutive two-electron transfers. NfsA has potential applications in the biodegradation of nitroaromatic environment pollutants, e.g. explosives, and is also of interest for the anticancer strategy gene-directed enzyme prodrug therapy. However, the catalytic mechanism of NfsA is poorly characterized. Here we examined the NADPH-dependent reduction of quinones (n = 16) and nitroaromatic compounds (n = 12) by NfsA. We confirmed a general "ping-pong" reaction scheme, and preliminary rapid reaction studies of the enzyme reduction by NADPH showed that this step is much faster than the steady-state turnover number, i.e., the enzyme turnover is limited by the oxidative half-reaction. The reactivity of nitroaromatic compounds (log kcat/Km) followed a linear dependence on their single-electron reduction potential (E17), indicating a limited role for compound structure or active site flexibility in their reactivity. The reactivity of quinones was lower than that of nitroaromatics having similar E17values, except for the significantly enhanced reactivity of 2-OH-1,4-naphthoquinones, consistent with observations previously made for the group B nitroreductase of Enterobacter cloacae. We present evidence that the reduction of quinones by NfsA is most consistent with a single-step (H-) hydride transfer mechanism.
authors.affiliation: Institute of Biochemistry of Vilnius University, Mokslininkų 12, LT-08662 Vilnius, Lithuania.
authors.forename: Benjaminas
authors.initials: B
authors.lastname: Valiauga
authors.affiliation: Victoria University of Wellington, School of Biological Sciences, Kelburn Parade, New Zealand.
authors.forename: Elsie M
authors.initials: EM
authors.lastname: Williams
authors.affiliation: Victoria University of Wellington, School of Biological Sciences, Kelburn Parade, New Zealand.
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
authors.affiliation: Institute of Biochemistry of Vilnius University, Mokslininkų 12, LT-08662 Vilnius, Lithuania. Electronic address: narimantas.cenas@bchi.vu.lt.
authors.forename: Narimantas
authors.initials: N
authors.lastname: Čėnas
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D004579:Electron Transport; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry; D009601:Nitroreductases / Q000737:chemistry*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011809:Quinones / Q000737:chemistry*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D011809:Quinones; D009249:NADP; C116053:NfsA protein, E coli; D009601:Nitroreductases; D009584:Nitrogen; D010100:Oxygen
keywords: Bioreductive activation; CB-1954; NfsA; Nitroaromatic compounds; Quinones