Multiscale machine learning molecular mechanics for mechanism and stereoselectivity of Diels-Alderase catalysis

Abstract Enzymes catalyze complex chemical transformations with remarkable efficiency and selectivity, yet their atomistic mechanisms remain challenging to capture because conventional simulations trade accuracy for efficiency. Here we introduce a reactive machine learning/molecular mechanics (ML/MM) framework that bridges quantum chemistry with long-timescale sampling, enabling direct exploration of enzymatic transition states and free-energy landscapes. Coupled with metadynamics, this approach achieves nanosecond sampling of bond-forming reactions and quantitatively predicts activation barriers, mutational effects, and stereoselectivity. Applied to Diels-Alderases, the framework not only reproduces experimental activity and endo / exo preferences with sub-kcal mol −1 accuracy but also uncovers how pathway dynamics and local electrostatics preorganize substrates for selective outcomes. By uniting reactivity, conformational dynamics, and predictive power, this work establishes reactive ML/MM as a broadly applicable strategy for mechanistic enzymology and a foundation for the rational design of new biocatalysts.