Abstract
Cytochrome P450 monooxygenases (P450s) are promising versatile oxidative biocatalysts. However, the practical use of P450s in vitro is limited by their dependence on the co-enzyme NAD(P)H and the complex electron transport system. Using H2O2 simplifies the catalytic cycle of P450s; however, most P450s are inactive in the presence of H2O2. By mimicking the molecular structure and catalytic mechanism of natural peroxygenases and peroxidases, an artificial P450 peroxygenase system has been designed with the assistance of a dual-functional small molecule (DFSM). DFSMs, such as N-(ω-imidazolyl fatty acyl)-l-amino acids, use an acyl amino acid as an anchoring group to bind the enzyme, and the imidazolyl group at the other end functions as a general acid-base catalyst in the activation of H2O2. In combination with protein engineering, the DFSM-facilitated P450 peroxygenase system has been used in various oxidation reactions of non-native substrates, such as alkene epoxidation, thioanisole sulfoxidation, and alkanes and aromatic hydroxylation, which showed unique activities and selectivity. Moreover, the DFSM-facilitated P450 peroxygenase system can switch to the peroxidase mode by mechanism-guided protein engineering. In this short review, the design, mechanism, evolution, application, and perspective of these novel non-natural P450 peroxygenases for the oxidation of non-native substrates are discussed.
Highlights
Cytochrome P450 monooxygenases (P450s) (CYPs or P450s), a broad class of heme-containing enzymes, play important roles in drug metabolism, detoxification of xenobiotics, and steroid biosynthesis [1]
It has become clear that to use the shunt pathway of P450s efficiently, two points should be met: (1) a basic group located on the distal side of the heme center is necessary; and (2) the basic group should be placed at a suitable position to ensure that this residue plays the role as an acid-base catalyst efficiently
The dual-functional small molecule (DFSM)-facilitated P450BM3 peroxygenase appears to open a new avenue for the key demethylation step in the bioconversion of lignin, it is still restricted by low turnover number (TON) and narrow substrate scopes
Summary
Cytochrome P450s (CYPs or P450s), a broad class of heme-containing enzymes, play important roles in drug metabolism, detoxification of xenobiotics, and steroid biosynthesis [1]. These enzymes are ubiquitous in nature, being found in animals, plants, bacteria, fungi, and other organisms [2]. P450s have potential use in the catalytic monooxygenation of various organic substrates, including aliphatic and aromatic compounds, alkenes, and compounds containing heteroatoms such as nitrogen and sulfur [3–8]. Researchers have constructed a substrate engineering approach to improve the acceptance and/or the stereo-/regioselectivity of non-native substrates of P450s by introducing protecting/anchoring/directing groups to Antioxidants 2022, 11, 529. The design, construction, mechanism, and catalytic application of the DFSM-facilitated P450 peroxygenases are reviewed, and current issues and future perspectives are discussed
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