Heterologous Overexpression and Biochemical Characterization of a Nitroreductase from Gluconobacter oxydans 621H

Mol Biotechnol. 2016 Jun;58(6):428-40. doi: 10.1007/s12033-016-9942-1.

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.

Keywords: CB1954; Nitroaromatic compound; Nitroreductase; Reduction.

MeSH terms

  • Aziridines / metabolism
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Gluconobacter oxydans / enzymology*
  • Gluconobacter oxydans / genetics
  • Kinetics
  • NADP / metabolism
  • Nitrobenzenes / metabolism
  • Nitroreductases / genetics*
  • Nitroreductases / metabolism*
  • Substrate Specificity
  • Trinitrotoluene / metabolism

Substances

  • Aziridines
  • Bacterial Proteins
  • Nitrobenzenes
  • Trinitrotoluene
  • NADP
  • tretazicar
  • nitrobenzene
  • Nitroreductases