Cytochrome P450 monooxygenases are a superfamily of oxidoreductase enzymes with heme iron as their active center. They play a vital role in the synthesis and metabolism of endogenous and exogenous substances within organisms. These enzymes are highly versatile biocatalysts with great potential for synthetic applications, attracting attention from researchers in synthetic chemistry and synthetic biology fields. However, the catalytic functionality of cytochrome P450 monooxygenases relies on coenzymes NAD(P)H and reductase proteins, which limits their catalytic applications outside of cellular systems.

To address the above-mentioned issue, Dr. Cong Zhiqi and his research group at the One-Carbon Biotechnology Research Center have simulated the catalytic mechanisms of natural peroxygenases and peroxidases. They have designed a class of dual-functional small molecules (DFSM) with acylated amino acids as anchoring groups and imidazole groups as catalytic groups. These DFSMs achieve the cooperative activation of hydrogen peroxide by the heme active center of P450, thereby transforming P450 monooxygenases into peroxygenase-like catalysts. By combining protein engineering, they have provided a unique solution to the challenging oxidation of carbon-hydrogen bonds (Angew. Chem. Int. Ed., 2018, 7628; 2022, e202215088; ACS Catal, 2019, 7350; 2021, 8449; Chem. Sci., 2021, 6307; J. Am. Chem. Soc., 2023, 5506).

However, the further practical application of this peroxygenase system is still limited by certain factors. Firstly, DFSMs exhibit very low affinity for P450, requiring more than 1000-fold excess amounts in the catalytic process. Secondly, the catalytic groups are currently limited to imidazole groups.

To this end, researchers studied the crystal structure of the complex formed by a typical DFSM, N-(ω-imidazolyl)-alkanoyl-L-phenylalanine (Im-C6-Phe), with P450BM3. They were inspired by the double conformational changes observed in DFSMs and hypothesized that by combining two amino acids to form a dipeptide as an anchoring group, they could obtain an artificial cofactor with high affinity, thus constructing a dual-center artificial peroxygenase. Among the 73 synthesized dipeptides, N-(ω-imidazolyl)-alkanoyl-L-amino acid-L-amino acid (Im-DFSM-dipeps), a series of DFSMs with significantly improved affinity (Kd ~ 10^-8 M) were screened, approaching the affinity of some natural enzymes/cofactors such as flavin-dependent enzymes. The loading of DFSM relative to P450BM3 was reduced from 1000 equivalents to 2 equivalents. Subsequently, the crystal complexes of the P450BM3 F87A variant with six Im-DFSM-dipeps were elucidated to explain the increased binding affinity. Taking Im-C6-Phe-Phe as an example, its carboxyl end directly formed hydrogen bonds with Tyr51, Arg47, Ala72, Gln73, and Ala74, while forming water-mediated hydrogen bond networks with Leu437, Ser72, and the carboxylic end of the heme via water molecules. Additionally, the two benzyl residues bind to two hydrophobic pockets composed of residues L20, Pro25, Val26, Leu29, Met185, and Leu188 (in yellow) as well as Leu17, Leu20, Phe42, Ala44, and Arg47 (in orange). The strong hydrogen bonding network and hydrophobic interactions synergistically anchor the Im-C6-Phe-Phe firmly on the enzyme, allowing the imidazole group to form hydrogen bonds with axial water molecules and remain in the appropriate position relative to the iron. Similar binding conformations were observed in other crystal complexes. Finally, the authors synthesized two series of novel dipeptide dual-functional molecules, replacing the imidazole with pyridyl and amine groups as acid-base catalytic groups. They found that the pyridyl-based DFSMs and amine-based DFSMs exhibited different substrate preferences and selectivities compared to the imidazole-based DFSM. This study provides a strategy for constructing dual-center artificial peroxygenases by anchoring editable cofactor-like modules as cooperative catalytic centers, thereby enhancing the catalytic multifunctionality of P450 enzymes.

Recently, the relevant work was published as a hot paper and cover article in the prestigious international journal Angewandte Chemie International Edition. Qin Xiangquan, a jointly trained doctoral student from Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) and Yanbian University, and Dr. Jiang Yiping from QIBEBT are the co-first authors of the paper. The corresponding authors are Dr. Cong Zhiqi from QIBEBT and Prof. Jin Longyi from Yanbian University. This research was supported by the National Natural Science Foundation, the National Key Research and Development Program, and the QIBEBT's Strong Foundation Plan, among others. (Text/Image: Qin Xiangquan, Jiang Yiping, Cong Zhiqi)

Original article link: https://doi.org/10.1002/anie.202311259

https://onlinelibrary.wiley.com/doi/10.1002/anie.202315458

　　Qin, X.#, Jiang, Y.#, Yao, F., Chen, J., Kong, F., Zhao, P., Jin, L.,* and Cong, Z.* Anchoring a structurally editable proximal cofactor-like module to construct an artificial dual-center peroxygenase. Angew. Chem. Inte. Ed. 2023, e202311259.