Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.
We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.
Zenno
S
S
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Japan.
Koike
H
H
Kumar
A N
AN
Jayaraman
R
R
Tanokura
M
M
Saigo
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000737:chemistry; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D009583:Nitrofurazone / Q000378:metabolism; D009601:Nitroreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D005486:Flavin Mononucleotide; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; D009583:Nitrofurazone