Probing Catalytic Residues Responsible for Primer Substrate Reactivity in a Versatile cis -Geranylgeranyl Diphosphate Synthase

Abstract Cis-isoprenyl diphosphate synthases (cis-IDSs) catalyze the synthesis of polyisoprenyl diphosphates with varying chain lengths and stereochemistry, yet the molecular mechanism underlying their substrate selectivity remains elusive. Here, we investigated VoDN3985, a cis-IDS from Viburnum odoratissimum, by combining mutagenesis, conditioned molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations. Conditioned MD simulations revealed substrate-dependent differences in geometric preorganization, with geranyl diphosphate (GPP) showing the highest prereactive fraction and farnesyl diphosphate (FPP) sampling more idealized allylic torsional geometries than dimethylallyl diphosphate (DMAPP). Active-site substitutions at Asn64 and His79 expanded primer tolerance to enable DMAPP utilization while preserving concerted substitution pathways in QM/MM profiles. Crucially, selectivity shift from altered configuration-dependent reaction energetics rather than barrier reductions. By decoupling dynamic conformational preorganization from intrinsic reaction-path energetics, this study provides actionable mechanistic principles for rationally engineering cis-IDS substrate selectivity.