Protein fusion strategies for a multi-component Rieske oxygenase

ABSTRACT Rieske oxygenases (ROs) are enzyme systems involved in microbial biodegradation or late-stage modifications during natural product biosynthesis. A major obstacle to working with ROs is their dependence on multi-component electron transfer chains (ETCs). Thereby, electrons from NAD(P)H are shuttled directly via a reductase (Red) or indirectly via an additional ferredoxin (Fd) to a terminal oxygenase (Oxy) for oxygen activation and subsequent substrate conversion. The present work evaluates potential fusion strategies to simplify the ETC of the three-component cumene dioxygenase (CDO) from Pseudomonas fluorescence . In in vitro reactions, the fusion of CDO-Red to CDO-Fd is the most suitable for activation of CDO-Oxy with product formation of approximately 22 mM (72 % conversion). Furthermore, protein fusion to CDO-Oxy was found to be feasible, highlighting the versatility of the redox partner fusion approach. Overall, this study aims to contribute to the research field of ROs by providing a promising strategy to simplify their multi-component nature.