The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield (R)-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting p-cresol, 2-methylnaphthalene or n-alkanes.

Anaerobic toluene degradation is initiated by adding toluene to fumarate via the glycyl radical enzyme benzylsuccinate synthase (BSS). We developed a recombinant production system producing active BSS and present data on the effects of some active-site mutants. A single substitution of Ile617 to Val inside a hydrophobic pocket of the active site leads to an extended substrate range of the variant, which includes conversion of m-xylene, but not of o- or p-xylene. Moreover, a mutation of the conserved Arg508, which participates in fumarate binding, to Lys or Gln decreases or eliminates BSS activity, proving its catalytic importance. Surprisingly, the Arg508Lys mutant gained the ability to synthesize a small amount of the product 3-benzyl-4-ketopentanoate from toluene and the fumarate analogue 3-acetyl acrylate. This study provides insight into substrate recognition by BSS and opens avenues for possible broader biotechnological applications in C–C bond formation.

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The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.