On the engineering of reductase-based-monooxygenase activity in CYP450 peroxygenases.

Abstract

Recent bioengineering of CYP450OleT shows that peroxide-based CYP450OleT can be converted to a reductase-based self-sufficient enzyme, which is capable of showing efficient hydroxylation and decarboxylation activity for a wide range of substrates. The so-generated enzyme creates several mechanistic puzzles: (A) as CYP450 peroxygenases lack the conventional acid-alcohol pair, what is the source of two protons that are required to create the ultimate oxidant Cpd I? (B) Why is it only CYP450OleT that shows the reductase-based activity but no other CYP members? The present study provides a mechanistic solution to these puzzles using comprehensive MD simulations and hybrid QM/MM calculations. We show that the fusion of the reductase domain to the heme-binding domain triggers significant conformational rearrangement, which is gated by the propionate side chain, which constitutes a new water aqueduct via the carboxylate end of the substrate that ultimately participates in Cpd I formation. Importantly, such well-synchronized choreographies are controlled by remotely located Tyr359, which senses the fusion of reductase and communicates to the heme domain via non-covalent interactions. These findings provide crucial insights and a broader perspective which enables us to make a verifiable prediction: thus, the catalytic activity is not only limited to the first or second catalytic shell of an enzyme. Furthermore, it is predicted that reinstatement of tyrosine at a similar position in other members of CYP450 peroxygenases can convert these enzymes to reductase-based monooxygenases.