Strategies for Substrate‐Regulated P450 Catalysis: From Substrate Engineering to Co‐catalysis

Abstract

Cytochrome P450 enzymes (P450s) catalyze the monooxygenation of various organic substrates. These enzymes are fascinating and promising biocatalysts for synthetic applications. Despite the impressive abilities of P450s in the oxidation of C−H bonds, their practical applications are restricted by intrinsic drawbacks, such as poor stability, low turnover rates, the need for expensive cofactors (e.g., NAD(P)H), and the narrow scope of useful non‐native substrates. These issues may be overcome through the general strategy of protein engineering, which focuses on the improvement of the catalysts themselves. Alternatively, several emerging strategies have been developed that regulate the P450 catalytic process from the viewpoint of the substrate. These strategies include substrate engineering, decoy molecule, and dual‐functional small‐molecule co‐catalysis. Substrate engineering focuses on improving the substrate acceptance and reaction selectivity by means of an anchoring group. The latter two strategies utilize co‐substrate‐like small molecules that either are proposed to reform the active site, thereby switching the substrate specificity, or directly participate in the catalytic process, thereby creating new catalytic peroxygenation capabilities towards non‐native substrates. For at least 10 years, these approaches have played unique roles in solving the problems highlighted above, either alone or in conjunction with protein engineering. Herein, we review three strategies for substrate regulation in the P450‐catalyzed oxidation of non‐native substrates. Furthermore, we address remaining challenges and potential solutions associated with these approaches.

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

“…The eukaryotic Class II P450s, with high substrate promiscuity, are generally not particularly suitable for synthetic and biotechnological applications due to their membrane-bound nature. To expand the substrate repertoire of prokaryotic soluble P450s, the strategy of "substrate engineering" has been practiced more often in recent years (14,103,104).…”

“…Distinct from typical substrate engineering using chemically or biologically modified substrates, Watanabe and his associates have systematically developed an atypical substrate engineering strategy, "decoy" substrate engineering, in which an inactive "dummy" substrate (decoy molecule) is used to trigger the P450-catalyzed reaction on the real nonnative substrate (103,104,111). Notably, there is no covalent linkage between the decoy and real substrates.…”

“…As an alternative strategy to protein engineering, the observed exquisite specificity and selectivity introduced by substrate engineering of P450 enzymes has highlighted the profound influence of the substrate-anchoring groups on the functional plasticity of P450s (103). Thus, this strategy has the potential to improve the synthetic utility of P450s.…”

“…Exciting new biotechnology approaches have contributed to breakthroughs in P450 system engineering for practical catalysis in the past decade (14,16,42,103). The multiple engineering strategies mentioned in this review have significantly improved the substrate scope, stability (60), catalytic efficiency (29), and reaction specificity (54) of P450s (Fig.…”

Molecular Dynamics Simulation and Kinetic Study of Fluoride Binding to V21C/V66C Myoglobin with a Cytoglobin-like Disulfide Bond

“…Protein design is able to not only reveal the structure-function relationship of native proteins, but also create artificial proteins with advanced functions [1][2][3][4][5][6][7][8][9][10][11][12]. This is especially the case for heme protein design, which has received much attention in the last few decades, and various approaches have been established for rational design, such as the introduction of non-heme metal ions and unnatural amino acids, and the use of heme mimics to act as an active site [1][2][3][4][5][6][7][8][9][10][11][12]. Importantly, computer modeling and molecular dynamics (MD) simulation play key roles in guiding the protein design [13][14][15][16][17][18].…”