The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions.

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The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH3) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO2. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultaneous adaptation; cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase, and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on toluene; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.

The initial step of anaerobic 4‐hydroxybenzoate and 3‐hydroxybenzoate degradation was studied in a denitrifying Pseudomonas sp. 4‐Hydroxybenzoate and 3‐hydroxybenzoate are converted into their coenzyme A (CoA) thioesters by two different specific coenzyme A ligases. 4‐Hydroxybenzoate‐CoA ligase (AMP‐forming) was purified 350‐fold. The ligase is active as a monomer of molecular mass 48 kDa, as determined by gel filtration and SDS/PAGE. At a pH optimum of 8.5, the apparent Km values for 4‐hydroxybenzoate, ATP, and coenzyme A are 37 μM, 77 μM, and 125 μM, respectively. The enzyme reacts specifically with 4‐hydroxybenzoate (100%) and 4‐aminobenzoate (30%). Other analogues of benzoate, notably 3‐ or 2‐hydroxybenzoate, are inactive, and 2,4‐dihydroxybenzoate and 2‐hydroxy‐4‐methylbenzoate act as competitive inhibitors (Ki= 1 μM). Polyclonal antibodies were raised and used in immunoblot assays to study the regulation of the expression of 4‐hydroxybenzoate‐CoA ligase. The ligase is synthesized when cells are grown anaerobically with 4‐hydroxybenzoate, phenol, or p‐cresol; phenol and p‐cresol are degraded via 4‐hydroxybenzoate. The enzyme is not present in cells grown aerobically with 4‐hydroyxbenzoate or anaerobically with benzoate or 4‐hydroxyphenylacetate.

The denitrifying Pseudomonas strain K172 was grown with a generation time of 6 h to a cell density of 0.4 g (dry weight) per liter with toluene and nitrate as substrates. We found that anaerobic cell suspensions oxidize [14C]toluene first to [14C]benzyl alcohol and subsequently to [14C]benzaldehyde. This proves that the methyl group of toluene is oxidized without molecular oxygen to a hydroxymethyl group.

A new pathway for the aerobic metabolism of 2-aminobenzoate which proceeds via 2-aminobenzoyl-CoA has recently been revealed in a Pseudomonas strain KB 740-. The enzyme catalyzing the first step, the formation of the coenzyme A (CoA) thioester of 2-aminobenzoate, is 2-aminobenzoate-CoA ligase. It was purified from cells aerobically grown with 2-aminobenzoate as sole carbon, energy, and nitrogen source and characterized. It is rather specific for 2-aminobenzoate, but activates also benzoate and fluorobenzoates. ATP was cleaved into AMP and pyrophosphate.

A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl‐CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl‐coenzyme A and 3‐hydroxybenzoyl‐coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851–4858)]. The initial reaction is catalyzed by benzoate‐CoA ligase (AMP‐forming), converting benzoate into benzoyl‐CoA. The next step is 3‐hydroxylation of benzoyl‐CoA to 3‐hydroxybenzoyl‐CoA catalyzed by a flavin‐nucleotide‐dependent monooxygenase, benzoyl‐CoA 3‐monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl‐CoA, but not benzoate, is hydroxylated. The protein is a monomer of Mr 65 000 and is induced when cells are grown aerobically with benzoate. 3‐Hydroxybenzoyl‐CoA is further hydroxylated para to the hydroxyl group affording 2,5‐dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3‐hydroxybenzoyl‐CoA 6‐monooxygenase, which is unspecific with respect to the pyridine nucleotide. Cells contain a second 6‐monooxygenase activity which acts on free 3‐hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl‐CoA, 3‐hydroxybenzoyl‐CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate.