Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and beta-oxidation of the first intermediate, (R)-(+)-benzylsuccinate

Anaerobic catabolism of toluene is initiated by addition of the methyl grou p of toluene to the double bond of a fumarate cosubstrate to yield the firs t intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-r adical enzyme benzylsuccinate synthase, as shown for the denitrifying bacte rium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. T he authors show here by experiments with cell extracts of toluene-grown T. aromatics that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, rever sible succinyl-CoA-dependent CoA-transferase. The product of the CoA-transf erase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl -CoA in extracts of toluene-grown cells, adding proof to the proposed anaer obic toluene-catabolic pathway. The stereochemical preferences of the enzym es catalysing formation and activation of benzylsuccinate have been analyse d. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzyl succinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of succinyl-CoA:ben zylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate a nd does not proceed with the (S)-(-)-enantiomer.