Issue 14, 2024

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.

Graphical abstract: On the engineering of reductase-based-monooxygenase activity in CYP450 peroxygenases

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Article information

Article type
Edge Article
Submitted
06 dec 2023
Accepted
06 mar 2024
First published
07 mar 2024
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2024,15, 5174-5186

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

S. Yadav, S. Shaik and K. D. Dubey, Chem. Sci., 2024, 15, 5174 DOI: 10.1039/D3SC06538C

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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