Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli.
It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
Hayashi
M
M
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Hasegawa
K
K
Oguni
Y
Y
Unemoto
T
T
D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D004790:Enzyme Induction; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D005486:Flavin Mononucleotide / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone); D010269:Paraquat / Q000494:pharmacology; D011808:Quinone Reductases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014812:Vitamin K / Q000494:pharmacology*
D009243:NAD; D014812:Vitamin K; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D010269:Paraquat