Find in Library

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 <i>N</i>-(ω-imidazolyl fatty acyl)-<span style="font-variant: small-caps;">l</span>-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.