Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate.

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 beta-oxidation of the benzylsuccinate formed is suggested.