BIOCHEMICAL AND GENETIC-CHARACTERIZATION OF BENZYLSUCCINATE SYNTHASE FROM THAUERA-AROMATICA - A NEW GLYCYL RADICAL ENZYME CATALYZING THE FIRST STEP IN ANAEROBIC TOLUENE METABOLISM

Toluene is anoxically degraded to CO2 by the denitrifying bacterium Th auera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate a s the first intermediate. We purified the enzyme catalysing this react ion, benzylsuccinate synthase (EC 4.1.99-), and studied its properties . The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 22 0 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels. The N-ter minal sequences of the alpha- and alpha'-subunits were identical, sugg esting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit. The composition of native enzyme therefore appears to be alpha(2) beta(2) gamma(2). A 5 kb Segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned a nd sequenced. The masses of the predicted gene products correlated exa ctly with those of the subunits, as determined by electrospray mass sp ectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enz ymes, particularly near the predicted radical site. The highest simila rity was observed with pyruvate formate lyases and related proteins. T he radical-containing subunit of benzylsuccinate synthase is oxygenoly tically cleaved at the site of the glycyl radical, producing the alpha '-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating prote in catalysing glycyl radical formation was found. The four genes for b enzylsuccinate synthase and the activating enzyme are organized as a s ingle operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-p romotor/polymerase system.