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

Masahiko Watanabe,Tatsuya Nishino,Koji Takio,Toshio Sofuni,Takehiko Nohmi

doi:10.1074/jbc.273.37.23922

Masahiko Watanabe, Tatsuya Nishino + Show 3 more

Open Access

https://doi.org/10.1074/jbc.273.37.23922

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Abstract

"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.

Highlights

“Classical” nitroreductase of Salmonella typhimurium is a flavoprotein that catalyzes the reduction of nitroaromatics to metabolites that are toxic, mutagenic, or carcinogenic
In 1990, we identified the nucleotide sequence of the gene encoding the nitroreductase of S. typhimurium and estimated that the enzyme is composed of 217 amino acids with a calculated Mr of 23,955 [21]
A base change mutation of T:A to G:C transversion at nucleotide 396 shown in the Fig. 1 of Ref. 21, which leads to a replacement of leucine 33 with arginine, was identified (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Enzymes—NADH, NADPH, NADPϩ, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Oriental Yeast (Tokyo, Japan). Direct DNA sequencing of the mutant nitroreductase gene of strain TA1538NR was carried out using polymerase chain reaction techniques described previously [13]. A fraction that eluted from a hydroxyapatite column showed a single band when analyzed by SDS-polyacrylamide gel electrophoresis followed by visualization with Coomassie Brilliant Blue This fraction was used as a source of the purified mutant nitroreductase. 1. Nucleotide and deduced amino acid sequences of the genes encoding the wild-type nitroreductase and the mutant nitroreductase of a strain S. typhimurium TA1538NR. Nucleotide and deduced amino acid sequences of the genes encoding the wild-type nitroreductase and the mutant nitroreductase of a strain S. typhimurium TA1538NR Nucleotide sequence of both DNA strands of the mutant gene was determined by direct sequencing after polymerase chain reaction amplification of the genes and by sequencing of the cloned genes. The incubated enzyme preparations were analyzed for their nitrofurazone-reductase activities

RESULTS

Crude extract

DISCUSSION

Flavins FMN FAD Riboflavin