Catalytic properties of phenol carboxylase In vitro study of CO₂: 4‐hydroxybenzoate isotope exchange reaction

Abstract

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.

Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium Thauera aromatica

“…Synthesis of both the phosphorylating and the carboxylating enzymes is strictly regulated. All activities were only present in cells grown anoxically on phenol and were lacking, e.g., in 4-hydroxybenzoate-grown cells (29,34). The phenol-carboxylating enzyme in T. aromatica does not belong to any group of the studied carboxylases, as it seems to proceed via a phosphorylated free intermediate, it is extremely oxygen sensitive and sensitive to radical-trapping agents (S. Breinig, M. Hügler, and G. Fuchs, unpublished results), and it is not dependent on biotin or thiamine diphosphate.…”

“…The process has been studied in the denitrifying bacterium Thauera aromatica (3,(32)(33)(34)63) (Fig. 1).…”

Hydroquinone degradation via reductive dehydroxylation of gentisyl-CoA by a strictly anaerobic fermenting bacterium

“…phenol (Lack et al 1991), aniline (Schnell and Schink 1991), o-cresol (Bisaillon et al 1991;Rudolphi et al 1991) or m-cresol (Roberts et al 1990). All these compounds are carboxylated in para-position to a hydroxyl or amino substituent.…”

“…For phenolic compounds, two different strategies are known: (i) Resorcinol and phloroglucinol degradation proceeds through direct reduction and subsequent hydrolytic cleavage of the aromatic ring (Kluge et al 1990;Brune and Schink 1992). (ii) Carboxylation, CoA-activation, and reductive dehydroxylation are the key reactions in anaerobic phenol degradation via benzoyl-CoA (Lack et al 1991;Gallert et al 1991).…”

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation

“…The cyclohexadiene reduction product(s) of benzoyl-CoA, and intermediates formed in subsequent reactions leading to alicyclic ring cleavage, all exist in CoAmodified form (23,30,43). Anaerobic growth substrates, such as phenol or toluene, that lack carboxyl groups may be modified by oxidation of a methyl substituent or by direct carboxylation to prepare a carboxylic acid intermediate suitable for CoA thioesterification (1,32).…”