Succinyl-CoA :(R)-benzylsuccinate CoA-transferase: an enzyme of the anaerobic toluene catabolic pathway in denitrifying bacteria

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first inte rmediate, which is further metabolized via beta -oxidation to benzoyl-coenz yme A (CoA) and succinyl-Coa. A specific succinyl-CoA:(R)-benzylsuccinate C oA-transferase activating (R)-benzylsuccinate to the CoA-thioester was puri fied and characterized from Thauera aromatica. The enzyme is fully reversib le and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some clo se chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccin ate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two s ubunits of similar amino acid sequences and similar sizes (44 and 45 kDa) i n an alpha (2)beta (2) conformation. Identity of the subunits with the prod ucts of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. T he deduced amino acid sequences resemble those of only two other characteri zed CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl- CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA- transferases. As suggested by kinetic analysis, the reaction mechanism of e nzymes of this family apparently involves formation of a ternary complex be tween the enzyme and the two substrates.