Computational Insights into an Enzyme-Catalyzed [4+2] Cycloaddition

[Image: see text] The enzyme SpnF, involved in the biosynthesis of spinosyn A, catalyzes a formal [4+2] cycloaddition of a 22-membered macrolactone, which may proceed as a concerted [4+2] Diels–Alder reaction or a stepwise [6+4] cycloaddition followed by a Cope rearrangement. Quantum mechanics/molecular mechanics (QM/MM) calculations combined with free energy simulations show that the Diels–Alder pathway is favored in the enzyme environment. OM2/CHARMM free energy simulations for the SpnF-catalyzed reaction predict a free energy barrier of 22 kcal/mol for the concerted Diels–Alder process and provide no evidence of a competitive stepwise pathway. Compared with the gas phase, the enzyme lowers the Diels–Alder barrier significantly, consistent with experimental observations. Inspection of the optimized geometries indicates that the enzyme may prearrange the substrate within the active site to accelerate the [4+2] cycloaddition and impede the [6+4] cycloaddition through interactions with active-site residues. Judging from partial charge analysis, we find that the hydrogen bond between the Thr196 residue of SpnF and the substrate C15 carbonyl group contributes to the enhancement of the rate of the Diels–Alder reaction. QM/MM simulations show that the substrate can easily adopt a reactive conformation in the active site of SpnF because interconversion between the C5–C6 s-trans and s-cis conformers is facile. Our QM/MM study suggests that the enzyme SpnF does behave as a Diels-Alderase.