Simulation-Guided Design of Cytochrome P450 for Chemo- and Regioselective Macrocyclic Oxidation

Abstract

Engineering high chemo-, regio-, and stereoselectivity is a prerequisite for enzyme usage in organic synthesis. Cytochromes P450 can oxidize a broad range of substrates, including macrocycles, which are becoming popular scaffolds for therapeutic agents. However, a large conformational space explored by macrocycles not only reduces the selectivity of oxidation but also impairs computational enzyme design strategies based on docking and molecular dynamics (MD) simulations. We present a novel design workflow that uses enhanced-sampling Hamiltonian replica exchange (HREX) MD and focuses on quantifying the substrate binding for suggesting the mutations to be made. This computational approach is applied to P450 BM3 with the aim to shift regioselectively toward one of the numerous possible positions during β-cembrenediol oxidation. The predictions are experimentally tested and the resulting product distributions validate our design strategy, as single mutations led up to 5-fold regioselectivity increases. We thus conclude that the HREX-MD-based workflow is a promising tool for the identification of positions for mutagenesis aiming at P450 enzymes with improved regioselectivity.

P450 Monooxygenases Enable Rapid Late‐Stage Diversification of Natural Products via C−H Bond Activation

“…Cytochrome P450 enzymes,, particularly BM3,, have been the subject of intense research computationally.. With macrocycles as the substrate, the study of Urlacher et al . in 2018 demonstrated the power of in silico engineering to impose control on the selectivity of P450 enzyme BM3 on the diterpenoid β ‐cembrenediol . In a previous study the Urlacher group had already investigated the BM3 on the β ‐cembrenediol ,.…”

“… The potential oxidation sites of β ‐cembrenediol on the left, and the three positions unselectively targeted by BM3 mutant V78A/F87A on the right …”

“…Using the mutant V78A/F87A as the starting template, a subsequent study computationally investigated what active site residue would significantly influence the enzyme's selectivity for 16 upon mutation . The strategy was based on the premise that the substrate's binding mode within the active site determines the selectivity of reaction.…”

“… β‐cembrenediol ( 16 ) is converted to epoxide 17 and hydroxylation products 18 and 19 by the BM3 mutant V78A/F87A . The percentage refers to the product distribution.…”

Selective C H bond functionalization with engineered heme proteins: new tools to generate complexity

“…In another demonstration, by combining large-scale MD simulations with sitesaturation mutagenesis, Dodani et al identified several mutations (His176Phe/Tyr/Trp) that completely redirect the regioselectivity of P450 TxtE-catalyzed nitration from the C4 to the C5 position of L-tryptophan [23•]. Additionally, there are also several studies that employ docking and MD simulation to identify important mutational hotspots for improving selectivity or substrate specificity of P450 hydroxylation [24,25,26].…”