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.

Results of Monte Carlo (MC) simulations for more than 200 nonnucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) representing eight diverse chemotypes have been correlated with their anti-HIV activities in an effort to establish simulation protocols and methods that can be used in the development of more effective drugs. Each inhibitor was modeled in a complex with the protein and by itself in water, and potentially useful descriptors of binding affinity were collected during the MC simulations. A viable regression equation was obtained for each data set using an extended linear response approach, which yielded r(2) values between 0.54 and 0.85 and an average unsigned error of only 0.50 kcal/mol. The most common descriptors confirm that a good geometrical match between the inhibitor and the protein is important and that the net loss of hydrogen bonds with the inhibitor upon binding is unfavorable. Other physically reasonable descriptors of binding are needed on a chemotype case-by-case basis. By including descriptors in common from the individual fits, combination regressions that include multiple data sets were also developed. This procedure led to a refined "master" regression for 210 NNRTIs with an r(2) of 0.60 and a cross-validated q(2) of 0.55. The computed activities show an rms error of 0.86 kcal/mol in comparison with experiment and an average unsigned error of 0.69 kcal/mol. Encouraging results were obtained for the predictions of 27 NNRTIs, representing a new chemotype not included in the development of the regression model. Predictions for this test set using the master regression yielded a q(2) value of 0.51 and an average unsigned error of 0.67 kcal/mol. Finally, additional regression analysis reveals that use of ligand-only descriptors leads to models with much diminished predictive ability.

The structure for the complex of nonnucleoside inhibitor TMC125 and HIV-1 reverse transcriptase has been determined and validated through computation of resistance profiles using Monte Carlo/free-energy perturbation calculations. The good quantitative agreement between the computed and experimental anti-HIV activities for TMC125, nevirapine, and efavirenz with wild-type RT and four common mutants (L100I, K103N, Y181C, and Y188L) confirms the correctness of the predicted structure and provides insights into the improved potency of this novel NNRTI. The blue shading in the figure indicates basic residues.

Chorismate mutase (CM) is an enzyme that catalyzes the Claisen rearrangement of chorismate to prephenate. In a recent effort to understand the basis for catalysis by CM, Kienhöfer and co-workers (J. Am. Chem. Soc. 2003, 125, 3206-3207) reported results on the mutation of Arg90 in Bacillus subtilis CM (BsCM) to citrulline (Cit), an isosteric but neutral arginine analogue. An ca. 10(4)-fold decrease in kcat or 5.9 kcal/mol increase in the free-energy barrier (ΔG(‡)) for the overall catalysis was observed upon mutation. In this work, attention is turned to determining the key factors that contribute to the reduced catalytic efficiency of Arg90Cit BsCM. Using a combined QM/MM Monte Carlo/Free-Energy Perturbation method, a ΔΔG(‡) value of 3.3 kcal/mol is obtained. The higher free-energy barrier for the mutant is exclusively related to inferior stabilization of the TS, particularly one of its carboxylate groups, by neutral Cit. In addition, the reaction becomes 2.0 kcal/mol more exergonic. As BsCM is limited by product release, this step contributes to the remainder of the 10(4)-fold decrease in the rate constant in going from Arg90 to Cit.