Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.
Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.
Hensgens
C M
CM
Department of Microbiology, University of Groningen, The Netherlands.
Hagen
W R
WR
Hansen
T A
TA
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001594:Benzyl Viologen / Q000378:metabolism*; D003067:Coenzymes; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008667:Metalloproteins / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis*; D000090006:Molybdenum Cofactors; D011487:Protein Conformation; D011621:Pteridines / Q000032:analysis; D017421:Sequence Analysis; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013455:Sulfur / Q000032:analysis; D014414:Tungsten / Q000032:analysis*
D000445:Aldehyde Oxidoreductases; D001594:Benzyl Viologen; D003067:Coenzymes; D004791:Enzyme Inhibitors; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D011621:Pteridines; D013455:Sulfur; D014414:Tungsten; C022261:molybdopterin; C096451:benzylviologen-linked aldehyde oxidoreductase