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

Abstract

AbstractSelf‐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.