The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate. The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied. This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins. Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of 14 CO 2 and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation. Phenylphosphate carboxylation was restored when the 18-kDa subunit was added. The following reaction model is proposed. The 14 CO 2 exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO 2 from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate. Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate. The 54-and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis. They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique. The 18-kDa subunit belongs to a hydratase/ phosphatase protein family. Phenylphosphate carboxylase is a member of a new family of carboxylases/ decarboxylases that act on phenolic compounds, use CO 2 as a substrate, do not contain biotin or thiamine diphosphate, require K ؉ and a divalent metal cation (Mg 2؉ or Mn 2؉ ) for activity, and are strongly inhibited by oxygen.The anaerobic metabolism of phenol has been studied to some extent in the beta-proteobacterium Thauera aromatica. The two initial steps of the pathway consist of the phosphorylation of phenol to phenylphosphate and the carboxylation of phenylphosphate to 4-hydroxybenzoate (36-38) (Fig. 1). Both enzyme activities are induced in cells grown anoxically on phenol and nitrate and not in cells grown on 4-hydroxybenzoate, the product of this process. 4-Hydroxybenzoate is also an intermediate in the metabolism of p-cresol (53)Further metabolism of 4-hydroxybenzoate proceeds via benzoyl coenzyme A (benzoyl-CoA) in two steps (Fig. 1). A specific CoA ligase forms 4-hydroxybenzoyl-CoA (7, 25), which is reductively dehydroxylated to benzoyl-CoA by a molybdoflavo-iron-sulfur protein, 4-hydroxybenzoyl-CoA reductase (13,15,26). The electron donor is a 2-[4Fe/4S] ferredoxin which is reduced by 2-oxoglutarate-ferredoxin oxidoreductase (21). Benzoyl-CoA is a common intermediate in the metabolism of many aromatic compounds. It is reductively d...

In the denitrifying member of the ␤-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acylCoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoateCoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V max /K m, the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate.

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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.

Anaerobic ethylbenzene metabolism in the betaproteobacterium Aromatoleum aromaticum is initiated by anaerobic oxidation to acetophenone via (S)-1-phenylethanol. The subsequent carboxylation of acetophenone to benzoylacetate is catalyzed by an acetophenone-induced enzyme, which has been purified and studied. The same enzyme is involved in acetophenone metabolism in the absence of ethylbenzene. Acetophenone carboxylase consists of five subunits with molecular masses of 70, 15, 87, 75, and 34 kDa, whose genes (apcABCDE) form an apparent operon. The enzyme is synthesized at high levels in cells grown on ethylbenzene or acetophenone, but not in cells grown on benzoate. During purification, acetophenone carboxylase dissociates into inactive subcomplexes consisting of the 70-, 15-, 87-, and 75-kDa subunits (apcABCD gene products) and the 34-kDa subunit (apcE gene product), respectively. Acetophenone carboxylase activity was restored by mixing the purified subcomplexes. The enzyme contains 1 Zn 2؉ ion per ␣␤␥␦ core complex and is dependent on the presence of Mg 2؉ or Mn 2؉ . In spite of the presence of Zn in the enzyme, it is strongly inhibited by Zn 2؉ ions. Carboxylation of acetophenone is dependent on ATP hydrolysis to ADP and P i , exhibiting a stoichiometry of 2 mol ATP per mol acetophenone carboxylated. The enzyme shows uncoupled ATPase activity with either bicarbonate or acetophenone in the absence of the second substrate. These observations indicate that both substrates may be phosphorylated, which is consistent with isotope exchange activity observed with deuterated acetophenone and inhibition by carbamoylphosphate, a structural analogue of carboxyphosphate. A potential mechanism of ATP-dependent acetophenone carboxylation is suggested.