Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.

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Anaerobic phenol degradation has been shown to proceed via carboxylation of phenol to 4-hydroxybenzoate. However, in vitro the carboxylating enzyme was inactive with phenol; only phenylphosphate (phosphoric acid monophenyl ester) was readily carboxylated. We demonstrate in a denitrifying Pseudomonas strain that phenylphosphate is the first detectable product formed from phenol in whole cells and that subsequent phenylphosphate consumption parallels 4-hydroxybenzoate formation. These kinetics are consistent with phosphorylation being the first step in anaerobic phenol degradation. Various cosubstrates failed so far to act as phosphoryl donor for net phosphorylation of phenol in cell extracts. Yet, cells anaerobically grown with phenol contained an enzyme that catalyzed an isotope exchange between [U-14C]phenol and phenylphosphate. This transphosphorylation activity was anaerobically induced by phenol but was stable under aerobic conditions and required Mn2+ and polyethylene glycol. Activity was optimal at pH 5.5 and half-maximal with 0.6 mM Mn2+, 0.2 mM phenylphosphate, and 1 mM phenol. It is proposed that the phenol exchange/transphosphorylation reaction is catalyzed as partial reaction by an inducible phenol phosphorylating enzyme. The isotope exchange demands that a phosphorylated enzyme was formed in the course of the reaction, which might be similar to the phosphotransferase system of sugar transport.

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14C02 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 pmol 14C02 incorporated into 4-hydroxybenzoate x min-x mg-cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14C02 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates C 0 2 and 4-hydroxybenzoate and required K + and Mn2+. The actual substrate was C 0 2 rather than HCO,. The apparent K,,, values were 1 mM dissolved COz, 0.2 mM 4-hydroxybenzoate, 2 mM K', and 0.1 mM MnZ+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K' could be replaced to some extent by Rb', but not by Li+, Na', Cs+, or NH:; Mn2+ could be replaced to some extent by Fe2+ >Mg2+, Co2', but not by Ni2+, Zn2+, CaZ+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group : derivatives of 4-hydroxybenzoate with OH, CH3 or C1 substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDSjPAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenolgrown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.Phenolic compounds are important plant constituents and phenol is formed by the activity of microorganisms from a variety of natural and synthetic substrates [l]. In addition phenol is one of the most prominent ground water contaminants. The aerobic metabolism of phenol has been studied extensively; as in all pathways of aerobic aromatic metabolism, oxygenases initiate the degradation of phenol [2].Evidence has recently been presented that phenol is metabolized to C 0 2 by pure bacterial cultures in the absence of molecular oxygen [3] (Fig.

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