Toluene is degraded anoxically to CO2 by the denitrifying bacterium Thauera aromatica. Toluene first becomes oxidized to benzoyl‐CoA by O2‐independent reactions. Benzoyl‐CoA is then reduced to non‐aromatic products by benzoyl‐CoA reductase. We set out to study the reactions employed for the initial activation of toluene and its oxidation to the level of benzoate. Evidence is provided for a novel way of toluene degradation based on experiments with cell‐free extracts and with whole toluene‐grown cells: Cell‐free extracts oxidized [14C]toluene to [14C]benzoyl‐CoA via several radioactive intermediates. This reaction was strictly dependent on the presence of fumarate, coenzyme A and nitrate as electron acceptor; acetyl‐CoA and ATP were not necessary for the reaction. The first product formed in vitro was benzylsuccinate; (2H8)toluene was converted to (2H7)benzylsuccinate. Formation of benzylsuccinate from toluene was independent of coenzyme A and nitrate, but it required the presence of fumarate. Other tricarboxylic acid cycle intermediates were converted to fumarate in cell extracts and therefore could partially substitute for fumarate. [14C]Benzylsuccinate was oxidized further to [14C]benzoyl‐CoA and [14C]benzoate in cell extracts if coenzyme A and nitrate were present. No benzyl alcohol and benzaldehyde and no phenylpropionate could be detected as intermediates. In isotope trapping experiments with cell suspensions, two intermediates from [14C]toluene were detected, benzoate and benzylsuccinate. This corroborates the sequence of reactions deduced from in vitro experiments. A hypothetical degradation pathway for the anaerobic oxidation of toluene to benzoyl‐CoA via an initial addition of fumarate to the methyl group of toluene and following β‐oxidation of the benzylsuccinate formed is suggested.

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.

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Toluene and related aromatic compounds are anaerobically degraded by the denitrifying bacterium Thauera sp. strain K172 via oxidation to benzoyl-CoA. The postulated initial step is methylhydroxylation of toluene to benzyl alcohol, which is either a free or enzyme-bound intermediate. Cells grown with toluene or benzyl alcohol contained benzyl alcohol dehydrogenase, which is possibly the second enzyme in the proposed pathway. The enzyme was purified from benzyl-alcohol-grown cells and characterized. It has many properties in common with benzyl alcohol dehydrogenase from Acinetobacter and Pseudomonas species. The enzyme was active as a homotetramer of 160 kDa, with subunits of 40 kDa. It was NAD(+)-specific, had an alkaline pH optimum, and was inhibited by thiol-blocking agents. No evidence for a bound cofactor was obtained. Various benzyl alcohol analogues served as substrates, whereas non-aromatic alcohols were not oxidized. The N-terminal amino acid sequence indicates that the enzyme belongs to the class of long-chain Zn(2+)-dependent alcohol dehydrogenases, although it appears not to contain a metal ion that can be removed by complexing agents.