Further characterization of trimethylamine N-oxide reductase from Escherichia coli, a molybdoprotein.
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.
D002384:Catalysis; D003067:Coenzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008667:Metalloproteins; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D000090006:Molybdenum Cofactors; D009247:NADH, NADPH Oxidoreductases / Q000145:classification / Q000302:isolation & purification*; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011621:Pteridines / Q000032:analysis; D013050:Spectrometry, Fluorescence
D003067:Coenzymes; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009247:NADH, NADPH Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011621:Pteridines; C022261:molybdopterin; C021567:methylamine dehydrogenase