title: Heterologous Overexpression and Biochemical Characterization of a Nitroreductase from Gluconobacter oxydans 621H.
abstract: A NADPH-dependent and FMN-containing nitroreductase (Gox0834) from Gluconobacter oxydans was cloned and heterogeneously expressed in Escherichia coli. The purified enzyme existed as a dimer with an apparent molecular mass of about 31.4 kDa. The enzyme displayed broad substrate specificity and reduced a variety of mononitrated, polynitrated, and polycyclic nitroaromatic compounds to the corresponding amino products. The highest activity was observed for the reduction of CB1954 (5-(1-aziridinyl)-2,4-dinitrobenzamide). The enzyme kinetics analysis showed that Gox0834 had relatively low K m (54 ± 11 μM) but high k cat/K m value (0.020 s(-1)/μM) for CB1954 when compared with known nitroreductases. Nitrobenzene and 2,4,6-trinitrotoluene (TNT) were preferred substrates for this enzyme with specific activity of 11.0 and 8.9 μmol/min/mg, respectively. Gox0834 exhibited a broad temperature optimum of 40-60 °C for the reduction of CB1954 with a pH optimum between 7.5 and 8.5. The purified enzyme was very stable below 37 °C over a broad pH range of 6.0-10.0. These characteristics suggest that the nitroreductase Gox0834 may be a possible candidate for catalyzing prodrug activation, bioremediation, or biocatalytic processes.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. jplin@ecust.edu.cn.
authors.forename: Jinping
authors.initials: J
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D001388:Aziridines / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009578:Nitrobenzenes / Q000378:metabolism; D009601:Nitroreductases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014303:Trinitrotoluene / Q000378:metabolism
chemical_list: D001388:Aziridines; D001426:Bacterial Proteins; D009578:Nitrobenzenes; D014303:Trinitrotoluene; D009249:NADP; C100099:tretazicar; C036077:nitrobenzene; D009601:Nitroreductases
keywords: CB1954; Nitroaromatic compound; Nitroreductase; Reduction