Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744.
The gene encoding the major NAD(P)H-flavin oxidoreductase (flavin reductase) of the luminous bacterium Vibrio fischeri ATCC 7744 was isolated by using synthetic oligonucleotide probes corresponding to the N-terminal amino acid sequence of the enzyme. Nucleotide sequence analysis suggested that the major flavin reductase of V. fischeri consisted of 218 amino acids and had a calculated molecular weight of 24,562. Cloned flavin reductase expressed in Escherichia coli was purified virtually to homogeneity, and its basic biochemical properties were examined. As in the major flavin reductase in crude extracts of V. fischeri, cloned flavin reductase showed broad substrate specificity and served well as a catalyst to supply reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. The major flavin reductase of V. fischeri not only showed significant similarity in amino acid sequence to oxygen-insensitive NAD(P)H nitroreductases of Salmonella typhimurium, Enterobacter cloacae, and E. coli but also was associated with a low level of nitroreductase activity. The major flavin reductase of V. fischeri and the nitroreductases of members of the family Enterobacteriaceae would thus appear closely related in evolution and form a novel protein family.
Zenno
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S
Yokohama Research Center, Chisso Corporation, Japan.
Saigo
K
K
Kanoh
H
H
Inouye
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004755:Enterobacteriaceae / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase; D005798:Genes, Bacterial / Q000235:genetics*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*
D011994:Recombinant Proteins; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D009601:Nitroreductases; D010100:Oxygen