The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via paracarboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were Phenol is a natural substrate which is formed from a variety of natural compounds. Phenol arises from tyrosine by tyrosine phenol lyase, but phenol also arises during the degradation of many secondary phenolic plant constituents, notably in the course of the degradation of lignin and phenylpropanoid compounds. Besides phenol, there are many other phenolic compounds, both natural and synthetic ones. Their mineralization proceeds via completely different pathways, depending on whether oxygen is available or not. For instance, groundwater and landfills are free of oxygen. Therefore, anaerobic metabolism of phenolic compounds is of general interest, from both scientific and applied aspects.The initial steps in aerobic phenol metabolism are catalyzed by oxygenases. Phenol is oxidized to catechol (1,2-dihydroxybenzene) by phenol monooxygenases followed by oxygenolytic ring cleavage catalyzed by catechol dioxygenase. Hence, aerobic metabolism of phenol requires molecular oxygen for both ring hydroxylation and ring cleavage. In contrast to aerobic metabolism, anaerobic metabolism cannot rely on oxygen-and oxygenase-dependent steps. Therefore, anaerobic metabolism of phenol and related phenolic compounds promises unprecedented biochemistry. Anaerobic growth of pure cultures on phenol has been shown for sulfate-reducing (5), denitrifying (47,50,51), and iron-reducing (35) bacteria. The list of bacteria growing anaerobically with phenolic compounds is steadily growing (see references in reference 43). In all cases studied, anaerobic growth on phenol requires the presence of CO 2 (50); CO 2 is required as a cosubstrate for phenol carboxylation which results in the formation of 4-hydroxybenzoate. Phenol carboxylation has been known in chemistry for more than 100 years and is referred to as Kolbe-Schmitt carboxylation.Anaerobic phenol metabolism by pure cultures has been studied in some detail only in the denitrifying beta-proteobacterium Thauera aromatica (2, 3, 16, 24, 29-31, 43, 50, 51). It involves two initial steps (Fig.

Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as shown by transcription analysis. In contrast to T. aromatica, transcription of the pps-and ppc-like genes was induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14 CO 2 and 4-hydroxybenzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the Thauera counterparts and located elsewhere in the genome.Anaerobic phenol degradation is best understood in the facultatively anaerobic denitrifier Thauera aromatica (DSM6984). In this strain, phenol is initially converted to phenylphosphate by phenylphosphate synthase (Pps) with concomitant hydrolysis of ATP (5, 16, 28) (Fig. 1A). The ␣-and ␤-subunits of Pps resemble the central and N-terminal parts of the phosphoenolpyruvate synthase, respectively. The ␤-subunit contains the ATP-binding moiety of the enzyme and is thought to transfer a diphosphoryl group to a conserved histidine residue in the ␣-subunit (23). There, orthophosphate is released and the ␤-phosphate group of ATP is transferred to phenol. Both subunits are therefore required for phosphorylation. The ␥-subunit is dispensable. However, its presence stimulates the reaction severalfold (28).In the next step, phenylphosphate is carboxylated by the action of phenylphosphate carboxylase (Ppc), yielding 4-hydroxybenzoate (4-OHB) (15,17,29). The ␦-subunit of the enzyme shows similarities to proteins of the hydrolase/phosphatase family (29). It was suggested to bind phenylphosphate and to catalyze its dephosphorylation, a reaction that is exergonic and virtually irreversible. The resulting phenolate anion is carboxylated by the core enzyme composed of ␣-, ␤-, an...

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO 2 as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.

Anaerobic phenol metabolism was studied in three facultative aerobic denitrifying bacteria, Thauera aromatica, "Aromatoleum aromaticum" strain EbN1 (Betaproteobacteria), and Magnetospirillum sp. (Alphaproteobacterium). All species formed phenylphosphate and contained phenylphosphate carboxylase but not phenol carboxylase activity. This is in contrast to direct phenol carboxylation by fermenting bacteria. Antisera raised against subunits of the Thauera phenylphosphate synthase and phenylphosphate carboxylase partly cross-reacted with the corresponding proteins in the other species. Some unsolved features of phenylphosphate carboxylase were addressed in T. aromatica. The core sub-complex of this enzyme consists of three different subunits and catalyzes the exchange of (14)CO(2) with the carboxyl group of 4-hydroxybenzoate, but not phenylphosphate carboxylation. It was inactivated by oxygen or by the oxidizing agent thionin and fully reactivated under reducing conditions. The purified recombinant phosphatase subunit alone had only low phenylphosphate phosphatase activity in the absence of the other components. However, activity was strongly enhanced in the presence of the core enzyme resulting in phenylphosphate carboxylation. Hence, a tight interaction of the carboxylase subunits is required for dephosphorylation of phenylphosphate, which is coupled to the concomitant carboxylation of the produced phenolate to 4-hydroxybenzoate, thus preventing a futile cycle.