Neophytadiene is an important volatile organic compound found in tobacco; however, its biosynthesis pathway remains unclear. In this study, linalool dehydratase-isomerase (LinD) was first mined and rationally designed for converting phytol to neophytadiene, resulting in an increase of kcat/Km from 9.89 to 11.84 min–1 mM–1. Structural analysis revealed that reduced steric hindrance and enhanced pocket hydrophobicity in the triple mutant (W243A-F39Y-V292D) improved ligand accessibility and promoted water elimination. Metadynamics simulations demonstrated that phytol adopts a deeper binding conformation within chain E of the mutant enzyme, with its hydroxyl group positioned nearer to the catalytic residues C179 and H128. In addition, molecular dynamics simulations indicated that increased flexibility between subunits facilitates efficient substrate transport. This study elucidates an engineered enzymatic route for the synthesis of neophytadiene.
