Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution

J Biol Chem. 2001 Jan 26;276(4):2816-23. doi: 10.1074/jbc.M002617200. Epub 2000 Oct 16.


The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Crystallography
  • Cytosol / metabolism
  • DNA Mutational Analysis
  • Drug Resistance, Neoplasm*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli Proteins*
  • FMN Reductase
  • Flavin Mononucleotide / chemistry*
  • Flavoproteins / chemistry*
  • Flavoproteins / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NADH, NADPH Oxidoreductases / chemistry
  • NADP / metabolism
  • Nitroreductases / chemistry*
  • Nitroreductases / genetics
  • Oxidation-Reduction


  • Escherichia coli Proteins
  • Flavoproteins
  • NADP
  • Flavin Mononucleotide
  • FMN Reductase
  • NADH, NADPH Oxidoreductases
  • NfsA protein, E coli
  • Nitroreductases

Associated data

  • PDB/1BKJ
  • PDB/1F5V
  • PDB/1PGO