The promiscuous activity of the enzyme o-succinylbenzoate synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both density functional theory and semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylamino acids, with N-succinyl-R-phenylglycine being the best substrate. We investigated the molecular mechanisms for both reactions exploring the potential energy surface. Then, molecular dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.