PMID:11034992
| Citation |
Kobori, T, Sasaki, H, Lee, WC, Zenno, S, Saigo, K, Murphy, ME and Tanokura, M (2001) 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. 276:2816-23 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M002617200 |
| Keywords |
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 |
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