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.
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.
Kobori
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T
Departments of Applied Biological Chemistry and Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Sasaki
H
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Lee
W C
WC
Zenno
S
S
Saigo
K
K
Murphy
M E
ME
Tanokura
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D003461:Crystallography; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D009249:NADP / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction
D029968:Escherichia coli Proteins; D005420:Flavoproteins; D009249:NADP; D005486:Flavin Mononucleotide; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; C116053:NfsA protein, E coli; D009601:Nitroreductases