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

European journal of biochemistry (1996-06-15)
T Biegert, G Fuchs, J Heider
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Toluene, anhydrous, 99.8%
Sigma-Aldrich
Toluene, ACS reagent, ≥99.5%
Sigma-Aldrich
Toluene, suitable for HPLC, 99.9%
Sigma-Aldrich
Toluene, HPLC Plus, for HPLC, GC, and residue analysis, ≥99.9%
Sigma-Aldrich
Toluene, puriss. p.a., ACS reagent, reag. ISO, reag. Ph. Eur., ≥99.7% (GC)
Sigma-Aldrich
Toluene, Laboratory Reagent, ≥99.3%
Sigma-Aldrich
Toluene, suitable for HPLC, ≥99.9%