4-Methoxybenzoate Monooxygenase from Pseudomonas putida: Isolation, Biochemical Properties, Substrate Specificity, and Reaction Mechanisms of the Enzyme Components

Studies on the mechanism of biological degradation of lignin or lignin model substances by fungi showed that degradation of lignin down to vanillic acid followed the pathway involving successively the intermediates: α-guaiacyl glycerolconiferyl ether, 4-hydroxy-3-methoxyphenylpyruvic acid, or 4-hydroxy-3-methoxycinnamic acid and vanillin. This chapter describes purification of the components of 4-methoxybenzoate monooxygenase. The enzyme activity of the cell-free crude extract is not proportional to protein concentration in the assay mixture, especially at low protein concentrations. This behavior shows that 4-methoxybenzoate monooxygenase is a dissociable enzyme system—that is, it consists of several components. This is confirmed by the isolation of two components, a reductase and a dioxygen-activating protein. The reconstitution of these two components reveals full enzymatic activity. The chapter also describes the properties of putidamonooxin (PMO). PMO, the dioxygen-activating component of the 4-methoxybenzoate monooxygenase, has a molecular weight of 126,000, as derived from ultracentrifugation and gel filtration.