An Enzyme Reducing Adenosine 1N-Oxide in Escherichia coli, Amine N-Oxide Reductase

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Abstract
1) Enzymatic reduction of adenosine 1N-oxide by an extract of Escherichia coli was followed by measuring decrease of absorbance of the compound at 233 mμ and that of dithionite at 317 mμ in the presence of benzylviologen and sodium dithionite added as an electron donor system. 2) The enzyme was purified 23 fold from the crude extract by isoelectric precipitation, ammonium sulfate precipitation, DEAE- and phospho-cellulose chromatog-raphies and Sephadex G-200 gel filtration. 3) Three protein peaks with enzyme activity were found on Sephadex filtration of a preparation obtained using a slightly modified purification process. Their molecular weights were 640, 000 (PI), 320, 000 (PII), and 160, 000 (PIII). The purified preparation was identified with PIII. 4) The properties of the purified preparation (PIII) were investigated. The optimum pH was 5.0–5.5, the isoelectric point was pH 4.6, and the Km was 2 x 10−4 M, but substrate inhibition was observed at concentrations over 0.3 mM. The main product was identified as adenosine by paper chromatography. N-Oxide or α- and γ-picoline, nicotinic acid trimethylamine, and nitrite were reduced. One mole of hydrogen was consumed per mole of N-oxide in a system coupled with hydrogenase. FAD, FMN, or cytochrome c served as electron donors, but NADH or NADPH did not. The reaction was stimulated by Fe2+ or Mn2+ and inhibited by α,α'-dipyridyl or o-phenanthroline. 5) A PII preparation was obtained by Sephadex gel filtration from cells after induction by growth in the presence of trimethylamine N-oxide. The properties of PII were essentially similar to those of PII. A preparatian of PII' with less ability to reduce trimethylamine N-oxide was obtained from another active peak obtained by Sephadex filtration. Nitrite reductase [EC 1.7.99.3] was eluted in a different position from the enzyme reducing N-oxide. 6) Bands of activity obtained by disc electrophoresis on polyacrylamide gel were negatively stained with reduced benzylviologen and N-oxides. One band from normal cells and three bands from cells grown in the presence of trimethylamine N-oxide were found and attributed to PIII, PII, and Pll'. The same bands were negatively stained with the N-oxide of adenosine and trimethylamine. 7) Addition of nitrate to the culture medium decreased the activity of an extract of the cells for N-oxide reduction. Addition of trimethylamine N-oxide induced the enzyme remarkably, while addition of adenosine N-oxide tended to cause decrease in activity. The nitrite-reducing activity was not affected by adding these compounds. 8) It is concluded from these results that the enzyme reducing adenosine N-oxide differs from nitrite reductase and should be called tertiary amine N-oxide reductase.

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