ABSTRACT
The meta O-dealkylase of Pseudomonas fluorescens Tp has been resolved into two protein components, neither of which is a cytochrome. The substrate binding terminal oxidase has been purified and shown to be a non-haem iron protein of approximate molecular weight 118,000, consisting of two seemingly identical subunits, each of molecular weight 55,000. Binding of substrate by the terminal oxidase has been established by difference spectroscopy. The amino acid composition of the protein has also been determined. The NADH-dependent reductase of the system has been partly purified and appears to have a molecular weight of 80,000. The similarity between this and other bacterial O-dealkylases is discussed.
Subject(s)
Hydroxybenzoates/metabolism , Oxidoreductases, O-Demethylating/isolation & purification , Oxidoreductases/isolation & purification , Pseudomonas aeruginosa/enzymology , Vanillic Acid/metabolism , Amino Acids/analysis , Cell-Free System , Dealkylation , Iron/analysis , Isoelectric Point , Molecular Weight , Oxidoreductases, O-Demethylating/metabolism , Oxygen ConsumptionSubject(s)
Cytochromes/metabolism , Multienzyme Complexes/metabolism , Nocardia/enzymology , Oxidoreductases/metabolism , Pseudomonas fluorescens/enzymology , Benzoates/metabolism , Cell Fractionation , Cytochrome Reductases/metabolism , Dealkylation , Electron Transport , Methyltransferases/metabolism , Mixed Function Oxygenases/metabolism , Molecular Weight , Nocardia/metabolism , Oxygen Consumption , Oxygenases/metabolism , Pseudomonas fluorescens/metabolismSubject(s)
Cytochrome P-450 Enzyme System/isolation & purification , Nocardia/analysis , Oxidoreductases/analysis , Amino Acids/analysis , Cell-Free System , Chromatography, Gel , Chromatography, Ion Exchange , Cytochrome P-450 Enzyme System/analysis , Cytochrome P-450 Enzyme System/metabolism , Dealkylation , Electron Spin Resonance Spectroscopy , Electrophoresis, Polyacrylamide Gel , Ethers/metabolism , Hemeproteins/metabolism , Isoelectric Focusing , Molecular Weight , Nocardia/enzymology , Nocardia/metabolism , Oxidoreductases/metabolism , Oxygen Consumption , Phenols/metabolism , Protein Binding , Spectrophotometry, Ultraviolet , UltracentrifugationSubject(s)
Cytochrome P-450 Enzyme System/isolation & purification , Multienzyme Complexes/isolation & purification , Nocardia/enzymology , Oxidoreductases/isolation & purification , Ammonium Sulfate , Benzoates/metabolism , Cell Fractionation , Cell-Free System , Chemical Precipitation , Chromatography, DEAE-Cellulose , Chromatography, Gel , Cytochrome Reductases/metabolism , Dealkylation , Electrophoresis, Polyacrylamide Gel , Multienzyme Complexes/metabolism , NADH, NADPH Oxidoreductases/metabolism , Nocardia/growth & development , Nocardia/metabolism , Oxidoreductases/metabolism , Oxygen Consumption , SpectrophotometrySubject(s)
Benzoates/metabolism , Nocardia/metabolism , Oxidoreductases/metabolism , Pseudomonas/metabolism , Acetaldehyde/metabolism , Aldehyde Oxidoreductases/metabolism , Aldehydes/metabolism , Cell-Free System , Culture Media , Dealkylation , Formaldehyde/metabolism , Glucose/metabolism , NAD/pharmacology , NADP/pharmacology , Nocardia/enzymology , Nocardia/growth & development , Oxidation-Reduction , Oxygen Consumption , Pseudomonas fluorescens/enzymology , Pseudomonas fluorescens/growth & development , Pseudomonas fluorescens/metabolism , Species Specificity , Spectrophotometry , UltrafiltrationABSTRACT
1. Protocatechuate 3,4-oxygenase in the soluble part of a cell-free extract of Pseudomonas fluorescens (strain T) sedimented more rapidly than vanillate O-demethylase under specified conditions in a preparative ultracentrifuge. 2. The supernatant from this process contained vanillate O-demethylase and formaldehyde dehydrogenase, and when supplemented with NADH oxidized vanillate with an uptake of 1 mole of oxygen/mole of substrate and accumulation of protocatechuate. 3. This uptake was decreased to 0.5mole/mole of substrate in the presence of semicarbazide as trapping agent for formaldehyde. 4. Reasons are presented for the process of methyl group removal from vanillate being oxidative demethylation.
Subject(s)
Oxygenases , Pseudomonas/enzymology , Transferases , Cell-Free System , Formaldehyde , Manometry , NAD , Oxidation-Reduction , Ultracentrifugation , UltrasonicsABSTRACT
1. A cell-free system from Pseudomonas fluorescens catalysed the oxidative demethylation and subsequent ring-cleavage of vanillate, with uptake of 2.5 moles of oxygen/mole of substrate. 2. Demethylation involved absorption of 0.5 mole of oxygen/mole, and required reduced glutathione (GSH) and nucleotide (probably NADPH) as cofactors, with further possible requirements, the natures of which are discussed. 3. Incomplete evidence suggested that the aromatic ring was opened via protocatechuate and the appropriate oxygenase, with absorption of 1 mole of oxygen/mole of substrate, eventually yielding beta-oxoadipate. 4. The methyl group was removed sequentially as formaldehyde, formate and carbon dioxide, the steps catalysed respectively by formaldehyde dehydrogenase, which required GSH and NAD(+), and formate dehydrogenase. Each enzyme was cytochrome-linked and accounted for absorption of 0.5mole of oxygen/mole of substrate. 5. All enzymes except formate dehydrogenase, which was a cell-wall enzyme, resided in the soluble fraction of the extract. The demethylase could not be resolved because of unknown cofactor requirements.
