Bacterial attack on phenolic ethers: An enzyme system demethylating vanillic acid

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 β-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.