Determinants for Substrate Recognition in the Glycyl Radical Enzyme Benzylsuccinate Synthase Revealed by Targeted Mutagenesis

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.