Purification and characterization of wild-type and mutant "classical" nitroreductases of Salmonella typhimurium. L33R mutation greatly diminishes binding of FMN to the nitroreductase of S. typhimurium.
"Classical" nitroreductase of Salmonella typhimurium is a flavoprotein that catalyzes the reduction of nitroaromatics to metabolites that are toxic, mutagenic, or carcinogenic. This enzyme represents a new class of flavin-dependent enzymes, which includes nitroreductases of Enterobacter cloacae and Escherichia coli, flavin oxidoreductase of Vibrio fischeri, and NADH oxidase of Thermus thermophilus. To investigate the structure-function relation of this class of enzymes, the gene encoding a mutant nitroreductase was cloned from S. typhimurium strain TA1538NR, and the enzymatic properties were compared with those of the wild-type. DNA sequence analysis revealed a T to G mutation in the mutant nitroreductase gene, predicting a replacement of leucine 33 with arginine. In contrast to the wild-type enzyme, the purified protein with a mutation of leucine 33 to arginine has no detectable nitroreductase activities in the standard assay conditions and easily lost FMN by dialysis or ultrafiltration. In the presence of an excess amount of FMN, however, the mutant protein exhibited a weak but measurable enzyme activity, and the substrate specificity was similar to that of the wild-type enzyme. Possible mechanisms by which the mutation greatly diminishes binding of FMN to the nitroreductase are discussed.
Watanabe
M
M
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Nishino
T
T
Takio
K
K
Sofuni
T
T
Nohmi
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017886:Durapatite; D016972:Enterobacter cloacae / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006190:Haemophilus / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009174:Mycoplasma / Q000201:enzymology; D009601:Nitroreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000201:enzymology; D014733:Vibrio / Q000201:enzymology
D005486:Flavin Mononucleotide; D017886:Durapatite; D009601:Nitroreductases