flavin-adenine-dinucleotide and tungstate

The aldehyde dehydrogenase activity of the sulfate-reducing bacterium Desulfovibrio simplex strain DSM 4141 was characterized in cell-free extracts. Oxygen-sensitive, constitutive aldehyde dehydrogenase activity was found in cells grown on l(+)-lactate, hydrogen, or vanillin with sulfate as the electron acceptor. A 1.83- to 2.6-fold higher specific activity was obtained in cells grown in media supplemented with 1 microM WO42-. The aldehyde dehydrogenase in cell-free extracts catalyzed the oxidation of aliphatic (Km < 20 microM) and aromatic aldehydes (Km < 0.32 mM) using methyl viologen as the electron acceptor. Flavins (FMN and FAD) were also active and are proposed to be the natural cofactors, while no activity was obtained with NAD+ or NADP+. 185WO42- was incorporated in vivo into D. simplex; it was found exclusively in the soluble fraction (>/= 98%). Anionic-exchange chromatography demonstrated coelution of 185W with two distinct peaks, the first one containing hydrogenase and formate dehydrogenase activities, and the second one aldehyde dehydrogenase activity.

Topics: Aldehyde Dehydrogenase; Aldehydes; Desulfovibrio; Enzyme Activation; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Oxidation-Reduction; Radioisotopes; Subcellular Fractions; Tungsten; Tungsten Compounds

Quinaldic acid 4-oxidoreductase from Pseudomonas sp. AK-2 catalyses the hydroxylation of quinoline 2-carboxylic (quinaldic acid) to 4-hydroxyquinoline 2-carboxylic acid (kynurenic acid) with concomitant reduction of a suitable electron acceptor. An analogous hydroxylation in para-position relative to the N-heteroatom was only recently described for quinaldine 4-oxidoreductase (de Beyer & Lingens, 1993, Biol. Chem. Hoppe-Seyler 374, 101-110) and for quinaldic acid 4-oxidoreductase from Serratia marcescens 2CC-1 (Fetzner & Lingens, 1993, Biol. Chem. Hoppe-Seyler 374, 363-376). Quinaldic acid 4-oxidoreductase from Pseudomonas putida AK-2 was purified 78-fold to electrophoretic homogeneity with a recovery of 22%. The native enzyme (300 kDa) was composed of three subunits with molecular masses of 90, 34 and 20 kDa, indicating an alpha 2 beta 2 gamma 2 structure. Quinaldic acid 4-oxidoreductase contained FAD, molybdenum, iron and acid-labile sulfur in a ratio of 2:2:8:8. Molybdenum is probably associated with molybdopterin cytosine dinucleotide as organic part of the pterin molybdenum cofactor. The absorption spectrum of quinaldic acid 4-oxido-reductase exhibited the typical features of a molybdo-iron/sulfur-flavoprotein, namely, maxima at 274 nm, 340 nm and 450 nm, a shoulder at 550 nm, a ratio A280/A450 of 4.7 and a ratio A450/A550 of 3.5. The enzyme was susceptible to inactivation by methanol, sodium m-arsenite, p-hydroxymercuribenzoate, and potassium cyanide. Cyanide caused an alteration at 320 nm in the absorption spectrum, typical for the change in the coordination sphere of the molybdenum. Enzyme inactivated with cyanide was reactivated to 74% by incubation with sulfide. Thus, quinaldic acid 4-oxidoreductase possesses a monooxo-monosulfido-type molybdenum center.

Topics: Catalysis; Electrophoresis, Polyacrylamide Gel; Flavin-Adenine Dinucleotide; Iron; Isoelectric Point; Metalloproteins; Mixed Function Oxygenases; Molecular Weight; Molybdenum; Oxidoreductases; Pseudomonas; Pseudomonas putida; Quinolines; Spectrophotometry, Ultraviolet; Sulfur; Tungsten Compounds