Interactions of Substrates with a Purified 4‐Methoxybenzoate Monooxygenase System ( O ‐Demethylating) from Pseudomonas putida

The interaction of substrates with a 4‐methoxybenzoate O ‐demethylating enzyme system was studied by use of crude cell‐free extracts and also by the purified enzyme system. The two components of the enzyme system, an iron‐containing flavoprotein and an iron‐sulphur protein, were obtained in pure state from Pseudomonas putida grown on 4‐methoxybenzoate as the sole carbon source. The purified enzyme system requires NADH and oxygen as cofactors and acts on various substrates. The highest affinity is found for 4‐methoxybenzoate, but also N ‐methyl‐4‐aminobenzoate is demethylated and 4‐alkylbenzoates are hydroxylated at the side chain. The enzyme is rather specific for para ‐substituted benzoic acid derivatives whereas 3‐methoxybenzoate and 4‐hydroxy‐3‐methoxybenzoate are demethylated slowly. The enzyme is also able to hydroxylate the aromatic ring. This is shown by the isolation of 3,4‐dihydroxybenzoate as the hydroxylation product of 3‐hydroxy‐ or 4‐hydroxy‐benzoate, respectively. Studies on substrate binding and oxygen consumption with substrate analogues showed an absolute requirement for the carboxy group at the aromatic ring. Benzoic acid derivatives without a suitable CH‐bond uncouple oxygen uptake with a concomitant formation of hydrogen peroxide. Measurements of oxygen consumption indicate that the affinity towards oxygen is substrate dependent, probably due to steric alterations as a consequence of substrate binding.