Structure and site-directed mutagenesis of a flavoprotein from Escherichiacoli that reduces nitrocompounds - Alteration of pyridine nucleotide binding by a single amino acid substitution

The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) f rom Escherichia coli has been solved by the molecular replacement method at 1.7-Angstrom resolution. This enzyme is a homodimeric flavoprotein with on e 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 si milar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite de finite difference in the spatial arrangement of residues around the putativ e 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 calalyt ic center FMN is predicted as a determinant for NADPH binding. The R203A mu tant results in a 33-fold increase in the K-m value for NADPH indicating th at the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.