Fig. 4: Computational modelling of the evolved KS.

a, Arrangement of the active site in variants P7E and KS characterized from MD simulations in their holo states. The cavity surface is shown in purple. b, Representative structure obtained from restrained MD simulations of trans-β-methylstyrene (1) bound in a reactive NAC in the KS active site. c, Analysis of active-site residues from accumulated MD trajectories. Top: increased rigidification from holo to substrate-bound states. ΔRMSF describes the root-mean-square fluctuation (RMSF) measured for active-site residues along NAC substrate-bound MD simulations compared with holo state simulations (RMSF(substrate bound) – RMSF(holo)). The more negative the value of ΔRMSF, the more rigid (that is less mobile) the residue becomes after binding the substrate in the simulated NAC. Bottom: interaction energies of individual residues and substrate bound in a NAC conformation. MM-GBSA substrate–residue pair interaction energies describe the strength of the interactions between trans-β-methylstyrene and surrounding active-site residues along the NAC substrate-bound MD simulations. Orange boxes highlight major changes in the flexibility of residues (ΔRMSF) and relevant interactions occurring between substrate and active site residues (MM-GBSA). d, Calculated QM/MM energy profile (ΔG and ΔE; the KS-wat model includes an explicit water molecule in the QM region, see Supplementary Computational Part II for details) for the competing epoxidation and ketone formation pathways for trans-β-methylstyrene (1) catalysed by KS (see also Supplementary Fig. 41). The energies of the doublet and quartet electronic states are reported in kcal mol⁻¹. e, QM/MM optimized geometries for the key radical intermediate (KS-wat-Intl^q) and carbocation intermediate (KS-wat-Int2^q) formed in the KS active site. ΔΔG and key distances are given in kcal mol⁻¹ and ångstroms (Å), respectively.