A strain of Pseudomonus putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the 0-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components : an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase.The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently.The absolute spectrum of the oxidized reductase displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores.The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 41 5 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the velocity and the extent of reduction of the iron and labile-sulfur-containing monooxygenase by the NADH-dependent reductase.Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i e . , iron-containing and labile-sulfur-containing monooxygenases.From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed. The 4-methoxybenzoate 0-demethylase from Pseudomonas putidu is a fairly unspecific enzyme. In the presence of NADH and molecular oxygen, the aliphatic C-H bond as well as the aromatic ring are attacked [ll]. Studies on substrate binding and oxygen aromatic ring, rather than the side chains of the Abbreviations. CD, circular dichroism; EPR, electron paramagnetic resonance, 2, mean g-value = 1/3 (gx + g, + gz).

The interaction of substrates with a 4-methoxybenzoate 0-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 Pseudomoms 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 a t 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 a t 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.The biodegradation of aromatic 0-alkyl ethers in bacteria is generally initiated by monooxygenase systems requiring reduced pyridine nucleotides and oxygen. An activated form of oxygen attacks the &-carbon atom and the methyl group is oxidatively eliminated probably via an unstable semiacetalUnlike the well-known nonspecific 0-dealkylating system in mammalian liver microsomes [2,3], the bacterial monooxygenases have been found to exhibit rather high specificity for the phenolic ethers on which the bacteria were grown.Cartwright et al. [4-61 have reported the existence of 3-and 4-methoxybenzoate demethylases in cell extracts from Pseudomonas fluorescens and Pseudomonas strain P 6. These enzymes again are different by pH-optima, sensitivity to inhibitors and stability against dialysis from the 4-methoxybenzoate 0-demethylating system which we have isolated from Pseudomonas putida [ 11. Recent investigations by Ribbons [7,8] on 3-methoxybenzoic acid 0-demethylases from P. aeruginosa and P. testosteroni in agreement with the results of Cartwright and our findings indicate that these monooxygenases may be multienzyme systems with similar structures.We have highly purified and characterized an iron-containing flavoprotein and an iron-sulphur protein (EPR at g = 1.91) as components of the 0-demethylating enzyme system from P. putidu grown on 4-methoxybenzoate [9]. There is no indication for the participation of a cytochrome, although Cartwight et al. [iO,ii] have recently described cytochrome P450 and another heme component in 0-demethylase systems from Noeardia NH 1 and P. fluorescens Tp, respectively. ...

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Zusammenfassung: Die vorliegende Arbeit berichtet über die enzymatischen Eigenschaften einer löslichen /7-Methoxybenzoesäure (/?-Anissäure)-0-Demethylase aus Pseudomonas species. Die Enzymaktivität des zellfreien Rohextraktes konnte auf das sechsfache angereichert werden. Das Enzym benötigt NADH oder NADPH und Sauerstoff als Cosubstrat für die Demethylierungsreaktion.Es ist demnach eine Monooxygenase (mischfunktionelle Oxygenase). Das Enzym desalkyliert auch höhere Homologe von /?-Methoxybenzoesäure und hydroxyliert/7-Toluylsäure zu^-Carboxybenzylalkohol. Die Hydroxylierung der Alkylgruppe dürfte daher der erste Schritt der Desalkylierung sein. Enzyminhibitoren sind /?-Chlormercuribenzoat, Formaldehyd, KCN, Cu 2 ®-, Zn 2 ®-und Cd 2 ®-Ionen, jedoch nicht Summary: The properties of a p-anisate O-demethylase in cell-free extracts of Pseudomonas spec. The present paper describes the enzymatic properties of a soluble /7-methoxybenzoic acid (p-anisate) <9-demethylase from Pseudomonas species. An approximately sixfold increase in enzymatic activity from the cell-free extract was achieved. NADH or NADPH and oxygen serve as cosubstrates for the reaction, thus characterizing the enzyme as a monooxygenäse (mixed function oxygenase). The enzyme also dealkylates higher homologues of /?-methoxybenzoic * Ein Teil der Ergebnisse wurde von F.B. während eines Studienaufenthaltes im

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