Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

Abstract

We recently developed an artificial P450–H2O2 system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H2O2 system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability.

Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

“…We then applied the developed water tunnel engineering strategy to improve the catalytic efficiency of the DFSM-facilitated P450BM3 peroxygenase under low H 2 O 2 concentrations. This system typically requires high H 2 O 2 concentrations (up to 80 mM for some reactions) to achieve high catalytic activity, despite the presence of the DFSM, which serves as a general acid–base catalyst to promote peroxygenase activity in P450BM3. ,, The water tunnels in a P450BM3 crystal containing a common DFSM ( N -(ω-imidazolyl)-hexanoyl- L -phenylalanine; Im-C6-Phe) were analyzed, and key residues at the tunnel entrance were screened using indole hydroxylation as a model reaction (Figures A, Figure S8, Table S8). Two single mutants, D182G and L272S, were identified based on the double mutant F87G/T268 V (GV) of the P450BM3 heme domain; these variants reached 114% and 153% activities, respectively, relative to the parent GV variant (Figures C, Tables S9 and S10).…”

Anchoring a Structurally Editable Proximal Cofactor‐like Module to Construct an Artificial Dual‐center Peroxygenase

“…The K d value of Im-C6-Phe-Phe reached 3.1×10 À 7 M, which is 310-fold lower than that of Im-C6-Phe (9.6×10 À 5 M). [29] Among the examined Im-DFSM-dipeps, Im-C6-Phe(4CF3)-Tyr exhibited the strongest affinity for P450BM3 (K d = 1.4×10 À 8 M), which was higher than that of some natural organic cofactors for their host enzymes (Figure 4A). These results suggest that Im-DFSM-dipeps can bind more tightly to P450BM3 and stably act as the second auxiliary active center for H 2 O 2 activation, similar to the catalytic residue in HRP or UPO.…”

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

“…Well-tempered metadynamics (WT-MTD) is a powerful enhanced sampling method that flattens the free energy surface to accelerate the occurrence of the rare events by periodically adding the time-dependent biasing potential on the specific collective variables (CVs), − and it has been widely used in the investigation of ligand binding or unbinding, drug-target interaction, and conformational dynamics. − In this study, 200 ns WT-MTD was employed to identify ligand-binding states with their free energy profiles, using NAMD 2.13 . The distance of substrate oxidation sites from the heme iron of P450 enzymes is often used to determine the metabolite type, and the distance should be less than 5 Å. ,,, Thus, the CVs were set to the distance between the two oxidation sites (C1′ and C4) and the heme iron in this work: Fe–C1′ and Fe–C4. The bin size is 0.1 Å, and the simulation temperature is 310 K. The Gaussian hill and width were set to 1 kcal/mol and 0.8 Å, respectively.…”

“…37 The distance of substrate oxidation sites from the heme iron of P450 enzymes is often used to determine the metabolite type, and the distance should be less than 5 Å. 15,42,46,47 Thus, the CVs were set to the distance between the two oxidation sites (C1′ and C4) and the heme iron in this work: Fe−C1′ and Fe−C4. The bin size is 0.1 Å, and the simulation temperature is 310 K. The Gaussian hill and width were set to 1 kcal/mol and 0.8 Å, respectively.…”