Modeling of the Reaction Mechanism of Enzymatic Radical C–C Coupling by Benzylsuccinate Synthase

Abstract

Molecular modeling techniques and density functional theory calculations were performed to study the mechanism of enzymatic radical C–C coupling catalyzed by benzylsuccinate synthase (BSS). BSS has been identified as a glycyl radical enzyme that catalyzes the enantiospecific fumarate addition to toluene initiating its anaerobic metabolism in the denitrifying bacterium Thauera aromatica, and this reaction represents the general mechanism of toluene degradation in all known anaerobic degraders. In this work docking calculations, classical molecular dynamics (MD) simulations, and DFT+D2 cluster modeling was employed to address the following questions: (i) What mechanistic details of the BSS reaction yield the most probable molecular model? (ii) What is the molecular basis of enantiospecificity of BSS? (iii) Is the proposed mechanism consistent with experimental observations, such as an inversion of the stereochemistry of the benzylic protons, syn addition of toluene to fumarate, exclusive production of (R)-benzylsuccinate as a product and a kinetic isotope effect (KIE) ranging between 2 and 4? The quantum mechanics (QM) modeling confirms that the previously proposed hypothetical mechanism is the most probable among several variants considered, although C–H activation and not C–C coupling turns out to be the rate limiting step. The enantiospecificity of the enzyme seems to be enforced by a thermodynamic preference for binding of fumarate in the pro(R) orientation and reverse preference of benzyl radical attack on fumarate in pro(S) pathway which results with prohibitively high energy barrier of the radical quenching. Finally, the proposed mechanism agrees with most of the experimental observations, although the calculated intrinsic KIE from the model (6.5) is still higher than the experimentally observed values (4.0) which suggests that both C–H activation and radical quenching may jointly be involved in the kinetic control of the reaction.

New tricks for the glycyl radical enzyme family

“…Exit of the product may also be promoted by the change in geometry within the active site—so far no product bound structure has been obtained. Quantum mechanical modeling based on the structure supports this proposed mechanism and suggests that H-atom abstraction is the rate-limiting step (Szaleniec and Heider, 2016). …”

Hydrocarbons and Lipids: An Introduction to Structure, Physicochemical Properties, and Natural Occurrence

“…However, Jarling et al (2012) identified the stereoisomers of (1-methylpentyl)succinate formed from anaerobic activation of n-hexane by an anaerobically cultivated bacterial strain, leading to the assumption that no alkyl radical intermediate but rather a concerted mechanism is involved in that reaction. Activation of toluene might still proceed via a benzyl radical (Szaleniec and Heider 2016;Seyhan et al 2016).…”

Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria

“…In contrast, all currently investigated anaerobic toluene degraders initiate the pathway through formation of (R)-benzylsuccinate from toluene and fumarate in a radical addition reaction catalyzed by the glycyl radical enzyme benzylsuccinate synthase (BSS) (Fig. 1) (6)(7)(8). The same pathway was found in many bacteria of different physiological groups that couple to various electron acceptors in anaerobic respiration, such as nitrate, sulfate, iron(III), manganese(IV), or carbonate, or degrade toluene phototrophically (1,8).…”