title: Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate.
abstract: Anaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. The authors show here by experiments with cell extracts of toluene-grown T. aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible succinyl-CoA-dependent CoA-transferase. The product of the CoA-transferase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway. The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(-)-enantiomer.
authors.affiliation: Mikrobiologie, Institut für Biologie II, Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany1.
authors.forename: Christina
authors.initials: C
authors.lastname: Leutwein
authors.affiliation: Mikrobiologie, Institut für Biologie II, Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany1.
authors.forename: Johann
authors.initials: J
authors.lastname: Heider
mesh_terms: D000693:Anaerobiosis; D001565:Benzoates / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D013386:Succinates / Q000378:metabolism*; D020628:Thauera / Q000201:enzymology* / Q000254:growth & development; D014050:Toluene / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism*
chemical_list: D001565:Benzoates; D003470:Culture Media; D013386:Succinates; C030196:benzylsuccinate; D014050:Toluene; D014166:Transferases