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

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