Abstract
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.
Highlights
The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-Å resolution
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 Arg203 and Arg208 of the loop region between helices I and J in the vicinity of the calalytic center FMN is predicted as a determinant for NADPH binding
Summary
NfsA, the major oxygen-insensitive NADPH-dependent nitroreductase of Escherichia coli; NfsB, the minor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coli; FRP, an NADPH-dependent flavin reductase of V. harveyi; NOX, an NADH oxidase of T. thermophilus; NfrA1, an NADPH-dependent nitro/flavin reductase of B. subtilis; FRase I, the major NAD(P)H:FMN oxidoreductase of V. fischeri; r.m.s., root mean square. There is a clear difference in the substrate specificity between NfsA and FRP, NfsA shares ϳ51% amino acid sequence identity with FRP. We show that Arg203 found in the NfsA/FRP subfamily is important for NADPH binding
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