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.
Highlights
Hydrocarbons have been believed for a long time to be persistent against microbial degradation in the absence of oxygen, since 1986 many bacteria have been shown to degrade these compounds anaerobically [1,2,3]
The reaction was soon confirmed to represent the general mechanism of toluene degradation in all known anaerobic degraders [13], and the enzyme catalyzing this unusual reaction was identified as a new glycyl radical enzyme, benzylsuccinate synthase (BSS) [14,15,16,17,18]
Very similar initiation reactions by fumarate additions catalyzed by closely related glycyl radical enzymes have since been described for anaerobic degradation of xylenes, ethylbenzene, p-cresol, 2-methylnaphthalene, p-cymene, and even alkanes, which seem to be added to fumarate at their subterminal methylene groups
Summary
Hydrocarbons have been believed for a long time to be persistent against microbial degradation in the absence of oxygen, since 1986 many bacteria have been shown to degrade these compounds anaerobically [1,2,3]. This product radical will be quenched by hydrogen transfer from the thiol group of Cys493, and after re-establishing the stable glycyl radical state of BSS by hydrogen transfer from Gly829 to the thiyl radical of Cys493, the product may be released and new substrates bound ([1], Figure 1) This mechanistic model was previously evaluated by gas-phase DFT modeling for activation of toluene and the aliphatic hydrocarbon butane and appears to be thermodynamically plausible [29,30]. Binding preferences with even more closely matching values between (S)- and (R)-benzylsuccinate have been reported previously from Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculations with the artificial active site predicted from the homology model [31]
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