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 <i>R</i>-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 <i>syn</i> 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 <i>R</i>-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 <i>R</i>-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.