Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

Abstract

Self‐sufficient cytochromes P450 of the sub‐family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5‐SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2O2. The full‐length enzyme (CYP116B5‐fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5‐hd) and CYP116B5‐SOX. The catalytic activity of the three forms of the enzyme was studied using p‐nitrophenol as substrate, and adding NADPH (CYP116B5‐fl), H2O2 (CYP116B5‐hd), and sarcosine (CYP116B5‐SOX) as source of electrons. CYP116B5‐SOX performs better than CYP116B5‐fl and CYP116B5‐hd showing 10‐ and 3‐folds higher activity, in terms of p‐nitrocatechol produced per mg of enzyme per minute. CYP116B5‐SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.

Alternative Electron Sources for Cytochrome P450s Catalytic Cycle: Biosensing and Biosynthetic Application

“…The main construction approach for the optimization of the electron transfer chain on electrodes as an alternative electron source involves the reductase domain or flavin cofactors into CYP and immobilization of fused protein onto the electrode surface [1,[7][8][9]49]. Different approaches for the optimization of the electron transfer chain in cytochrome P450 electrochemical systems were proposed earlier [55][56][57][58][59][60][61][62][63][64][65][66].…”

“…The G. Gilardi group proposed the construction of effective electron transfer chains using the "Lego" approach, combining the heme domain of bacterial CYP102 A1 (BM3), CYP116B5 or CYP3A4 and the reductase domain of BM3 [58][59][60][61][62][63][64][65][66]. These constructs demonstrated enhanced efficiency in electrochemical systems.…”

Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study

“…Then, the latter can, through a stepwise or dynamically concerted radical rebound mechanism, immediately perform the hydroxylation reaction [ 20 , 21 ]. Therefore, without needing expensive electron donors, such as NADPH, used in the classical CYP450 catalysis, but simply by using hydrogen peroxide or peracids, products can be obtained with high catalytic performances [ 19 , 22 ].…”

Biocatalytic methane oxidation

“…A set of self-sufficient class VII P450s (primarily from the CYP116B subfamily, e.g., CYP116B2) have been observed to catalyze a wide variety of C−H activations across various substrates [25][26][27][28] . It has been proposed that these CYP116B enzymes hold promise as highly desirable biocatalysts for synthetically challenging reactions, including diverse C−H activations 29 .…”