Enzymes are highly specific for their native functions, however with advances in bioengineering tools such as directed evolution, several enzymes are being repurposed for the secondary function of contemporary significance. Due to the functional versatility, the Cytochrome P450 (CYP450) superfamily has become the ideal scaffold for such bioengineering. In the current study, using MD molecular dynamics) simulations and hybrid QM/MM (Quantum mechanics/molecular mechanics) calculations, we have studied the mechanism of spontaneous emergence of a secondary function due to a single site mutation in two plant CYP450 enzymes from the mint family. The MD simulations of WT (wild type) CYP71D18 and CYP71D13 enzymes and their variants show a crucial gating mechanism by aromatic dyad formed by Phe121 and Phe363 which regulates the substrate recognition. The QM/MM calculations reveal that the hydroxylation reactions at C3 and C6 positions in WT CYP71D18 and CYP71D13 enzymes as well as their variants follow a hydrogen atom transfer (HAT) followed by a single electron transfer (SET) mechanism, which is different from the typical rebound mechanism shown by most of the CYP450 enzymes.
Supporting material contains RMSD, RMSD, QMMM calculations for another snapshots and coordinates used in QMMM calculations.