Macrophomate Synthase: QM/MM Simulations Address the Diels−Alder versus Michael−Aldol Reaction Mechanism

Macrophomate synthase (MPS) of the phytopathogenic fungus Macrophoma commelinae catalyzes the transformation of 2-pyrone derivatives to the corresponding benzoate analogues. The transformation proceeds through three separate chemical reactions, including decarboxylation of oxalacetate to produce pyruvate enolate, two C−C bond formations between 2-pyrone and pyruvate enolate that form a bicyclic intermediate, and final decarboxylation with concomitant dehydration. Although some evidence suggests that the second step of the reaction catalyzed by MPS is a Diels−Alder reaction, definite proof that the C−C bond formations occur via a concerted mechanism is still required. An alternative route for formation of the C−C bonds is a stepwise Michael−aldol reaction. In this work, mixed quantum and molecular mechanics (QM/MM) combined with Monte Carlo simulations and free-energy perturbation (FEP) calculations were performed to investigate the relative stabilities of the transition states (TS) for both reaction mechanisms. The key results are that the Diels−Alder TS model is 17.7 and 12.1 kcal/mol less stable than the Michael and aldol TSs in the stepwise route, respectively. Therefore, this work indicates that the Michael−aldol mechanism is the route used by MPS to catalyze the second step of the overall transformation, and that the enzyme is not a natural Diels−Alderase, as claimed by Ose and co-workers (Nature 2003, 422, 185−189; Acta Crystallogr. 2004, D60, 1187−1197). A modified link-atom treatment for the bonds at the QM/MM interface is also presented.