Protein-protein interactions and antigenic relationships between the components of 4-methoxybenzoate monooxygenase and of benzene 1,2-dioxygenase from Pseudomonas putida.
The investigations presented in this paper were performed on two enzyme systems from Pseudomonas putida: (a) 4-methoxybenzoate monooxygenase, consisting of a NADH: putidamonooxin oxidoreductase and putidamonooxin, the oxygen-activating component, and (b) benzene 1,2-dioxygenase, a three-component enzyme system with an NADH: ferredoxin oxidoreductase, functioning together with a plant-type ferredoxin as electron-transport chain, and an oxygen-activating component similar to putidamonooxin in its active sites. The influence of temperature, ionic strength, and pH on the activities of 4-methoxybenzoate monooxygenase and of NADH: putidamonooxin oxidoreductase were investigated. The studies revealed that the activity of 4-methoxybenzoate monooxygenase is determined by the behaviour of the reductase. Spectroscopic measurements showed that the interaction between the two components of 4-methoxybenzoate monooxygenase influences the optical-absorption behaviour of one or both components. As a criterion for the affinity between the two components of 4-methoxybenzoate monooxygenase, the Km value of the reductase for putidamonooxin was determined and found to be 31 +/- 11 microM. Antibodies against both components of 4-methoxybenzoate monooxygenase were obtained from rabbits. The antibodies against putidamonooxin inhibited the O-demethylation reaction (up to 80%) and also the reduction of putidamonooxin by the reductase (up to 40%). The antibodies against putidamonooxin did not interact with the oxygen-activating component of benzene 1,2-dioxygenase. The electron-transport chains of 4-methoxybenzoate monooxygenase and benzene 1,2-dioxygenase could not be replaced by one another without a complete loss of enzyme activity.
D000818:Animals; D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010090:Oxidoreductases, O-Demethylating / Q000276:immunology / Q000378:metabolism*; D010100:Oxygen; D010101:Oxygen Consumption; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000276:immunology; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology* / Q000276:immunology; D011817:Rabbits; D013379:Substrate Specificity; D013696:Temperature
D000942:Antigens, Bacterial; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; D010105:Oxygenases; C022485:putidamonooxin; C019765:benzene 1,2-dioxygenase; C021562:4-methoxybenzoate monooxygenase (O-demethylating); D010100:Oxygen