Unravelling the Enantioselective Mechanism of Benzylsuccinate Synthase: Insights into Anaerobic Hydrocarbon Degradation through Multiscale Modeling and Microkinetics

Fumarate-adding
enzymes (FAE) are a subset of the glycyl
radical
enzyme superfamily involved in anaerobic hydrocarbon degradation.
Benzylsuccinate synthase (BSS) catalyzes the enantiospecific formation
of R-benzylsuccinate from toluene and fumarate, initiating
anaerobic toluene degradation. In this paper, we present a detailed
theoretical study of the reaction mechanism using classical molecular
dynamics and multiscale modeling (QM/MM). We describe the potential
energy surface of the reaction and confirm the previously postulated
mechanism. However, the multiscale character of our model allowed
us to elucidate the origins of several experimentally observed catalytic
phenomena, such as the inversion of the benzylic carbon configuration
upon C–C bond formation and the syn addition
of the abstracted H atom back to the benzylsuccinyl radical. The obtained
model is supported by microkinetic analysis and was able to explain
and quantitatively predict the strict R-enantioselectivity
of BSS, which is enforced predominantly by the dynamic kinetic behavior
of toluene in the active site, leading to over 40-times faster production
of the R-enantiomer, not by the binding orientation
of the fumarate. Our study contributes to the elucidation of the catalytic
processes catalyzed by BSS and its role in the bioremediation of hydrocarbon
pollutants.