PMID:3528139
| Citation |
Yamamoto, I, Okubo, N and Ishimoto, M (1986) Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein. J. Biochem. 99:1773-9 |
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| Abstract |
Escherichia coli trimethylamine N-oxide (TMAO) reductase I, the major enzyme among inducible TMAO reductases, was purified to homogeneity by an improved method including heat treatment, ammonium sulfate precipitation, and chromatographies on Bio-Gel A-1.5m, DEAE-cellulose, and Reactive blue-agarose. The molecular weight was estimated by gel filtration to be approximately 200,000. A single subunit peptide with a molecular weight of 95,000 was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme contained 1.96 atoms of molybdenum, 0.96 atoms of iron, 1.52 atoms of zinc, and less than 0.4 atoms of acid-labile sulfur per molecular weight of 200,000. The absorption spectrum of the enzyme showed a peak at 278 nm and a shoulder at 288 nm, but no characteristic absorption was found from 350 to 700 nm. A fluorescent derivative of molybdenum cofactor was found when the enzyme was boiled with iodine in acidic solution; its fluorescence spectra were almost the same as those of the form A derivative of molybdopterin found in sulfite oxidase. The molybdenum cofactor released from heated TMAO reductase I reconstituted nitrate reductase in the extracts of Neurospora crassa mutant strain nit-1 lacking molybdenum cofactor. Thus, TMAO reductase I contains molybdopterin, which is a common constituent of some molybdenum-containing enzymes. Some kinetic properties were also determined. |
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| Keywords |
Catalysis; Coenzymes; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Metalloproteins; Molecular Weight; Molybdenum/analysis; NADH, NADPH Oxidoreductases/classification; NADH, NADPH Oxidoreductases/isolation & purification; Oxidoreductases Acting on CH-NH Group Donors; Pteridines/analysis; Spectrometry, Fluorescence |
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