PURIFICATION AND SOME PROPERTIES OF INDUCIBLE TERTIARY AMINE N-OXIDE REDUCTASE FROM ESCHERICHIA-COLI

Formation of amine N-oxide reductase in E. coli was repressed by tungstate but not when molybdate was present. Four forms of this reductase with different electrophoretic mobilities on polyacrylamide gel were found by active staining using the supernatant obtained by the centrifugation at 17,000 x g of the extract from cells grown in the presence of trimethylamine AT-oxide; one of them was constitutive and the other three, including the most active, were inducible. The reductase found in the precipitate obtained by centrifugation at 100,000 X g was solubilized by treatment with Emulgen 810 and showed two forms, one of which corresponded to the most active form found in the supernatant. The reductase was purified from the supernatant to an almost homogeneous state by ammonium sulfate precipitation and DEAE-cellulose, Sephadex G-200, double DEAE-Sephadex A-25, and hydroxyapatite chro-matographies. Specific activity was increased about 100 fold and recovery was 1%. The enzyme corresponded to the most active form. The purified enzyme reduced various N-oxides of purine and pyridine derivatives as well as trimethylamine iV-oxide, hydroxylamine, chlorate, and bromate. The optimal pH's for trimethylamine JV-oxide, adenosine N-oxide, and hydroxylamine were 5.3, 6.0, and 6.0, and Km values 1.5 itim, 0.36 mM, and 53 mM, respectively. FMN and FAD, but not cytochrome c, served as electron donors beside benzyl and methyl viologen, though the reaction rates were about one tenth. The enzyme was activated by ferric ion, and severely inhibited by cupric ion. The spectrum showed a peak at 280-nm and a shoulder at 385 nm. Molecular weight was estimated to be 220,000 by Sephadex G-200 gel filtration. Isoelectric point was pH 5.2. Iron content was 1.8mol/mol. The enzyme was found to consist of at least two sorts of subunits, the molecular weights of which were found to be 60,000 and 80,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition is also presented. © 1979, by the Japanese Biochemical Society.