title: Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate.
abstract: 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.
authors.affiliation: Institut für Biologie II, Universität Freiburg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Biegert
authors.forename: G
authors.initials: G
authors.lastname: Fuchs
authors.forename: J
authors.initials: J
authors.lastname: Heider
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D002250:Carbon Radioisotopes; D002457:Cell Extracts; D005650:Fumarates / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D013386:Succinates / Q000378:metabolism*; D014050:Toluene / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D001565:Benzoates; D002250:Carbon Radioisotopes; D002457:Cell Extracts; D005650:Fumarates; D013386:Succinates; C030196:benzylsuccinate; D014050:Toluene; C037817:benzoyl-coenzyme A; C032005:fumaric acid; D019817:Benzoic Acid