Enzymes catalyzing [4+2] cycloadditions are involved in the formation of complex structures found in natural products, and play key roles in the control of stereochemistry. Fsa2 and Phm7 catalyze intramolecular [4+2] cycloaddition to form enantiomeric decalin scaffolds during the biosynthesis of HIV-1 integrase inhibitors, equisetin, and an opposite stereochemical homolog, phomasetin. Here, we solved the X-ray crystal structures of substrate-free Fsa2 and Phm7, and an inhibitor-bound Phm7 to

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... erstand the molecular basis underlying stereoselective cycloaddition reactions. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, which explain the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyze the stereoselective cycloaddition. This powerful combination of experimental and theoretical approaches highlights the distinct molecular mechanisms of enzyme-mediated [4+2] cycloaddition and its stereoselectivity.