flavin-adenine-dinucleotide and catechol

The purpose of this study was purification and characterization of phenol monooxygenase from Stenotrophomonas maltophilia strain KB2, enzyme that catabolises phenol and its derivatives through the initial hydroxylation to catechols. The enzyme requires NADH and FAD as a cofactors for activity, catalyses hydroxylation of a wide range of monocyclic phenols, aromatic acids and dihydroxylated derivatives of benzene except for catechol. High activity of this monooxygenase was observed in cell extract of strain KB2 grown on phenol, 2-methylphenol, 3-metylphenol or 4-methylphenol. Ionic surfactants as well as cytochrome P450 inhibitors or 1,4-dioxane, acetone and n-butyl acetate inhibited the enzyme activity, while non-ionic surfactants, chloroethane, ethylbenzene, ethyl acetate, cyclohexane, and benzene enhanced it. These results indicate that the phenol monooxygenase from Stenotrophomonas maltophilia strain KB2 holds great potential for bioremediation.

Topics: Benzene; Biodegradation, Environmental; Catechols; Cresols; Cytochrome P-450 Enzyme Inhibitors; Dioxanes; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Mixed Function Oxygenases; NAD; Phenol; Stenotrophomonas maltophilia; Substrate Specificity; Surface-Active Agents

The aim of this work was to investigate the hypothesis that catechol inhibits FADH -linked basal respiration in mitochondria isolated from rat liver homogenates. Moreover, catechol ability to induce peroxidation of biomolecules in liver nuclear fractions was also studied.. Rat liver homogenates were incubated with 1mM 1,2-dihydroxybenzene (catechol) at pH 7.4 for up to 30 minutes. After that, mitochondrial fractions were isolated by differential centrifugation. Basal oxygen uptake was measured using a Clark-type electrode after the addition of 10 mM sodium succinate. Nuclear fractions were incubated in the presence of 1 mM catechol for 17 hours at room temperature and the peroxidation of biomolecules was investigated by the reaction with thiobarbituric acid, which was determined spectrophotometrically at 535 nm.. Catechol induced a time-dependent partial inhibition of FADH -linked basal mitochondrial respiration, however this substance was unable to induce a direct peroxidation of biomolecules in hepatic nuclear fractions.. Catechol produced an inhibition of basal respiration associated to FADH2 in isolated liver mitochondria that could lead to cytotoxicity, ROS generation and cell death.

Topics: Animals; Catechols; Cell Nucleus; Cell Respiration; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Lipid Peroxidation; Mitochondria, Liver; Oxidative Stress; Oxygen Consumption; Rats; Rats, Wistar; Reactive Oxygen Species; Time Factors

A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group.

Topics: Amino Acid Sequence; Archaeoglobus fulgidus; Bacillus; Catalysis; Catechols; Chromatography, High Pressure Liquid; Cysteine; Dimerization; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Flavin-Adenine Dinucleotide; Kinetics; Mixed Function Oxygenases; Models, Chemical; Models, Molecular; Molecular Sequence Data; NAD; Phenol; Plasmids; Protein Binding; Recombinant Proteins; Sequence Homology, Amino Acid; Spectrophotometry; Temperature

Topics: Catechols; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Hydroxylation; Mixed Function Oxygenases; NAD; Oxygen Isotopes; Research; Salicylates; Sulfonic Acids

Topics: Benzoquinones; Catechols; Chelating Agents; Cyanides; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Oxidoreductases; Quinacrine; Quinones; Research; Vegetables