PhD defence M.E. (Michael) Runda

Rieske oxygenases outside a nutshell

Enzyme-catalyzed oxyfunctionalization reactions are of great interest in synthetic chemistry due to their potential for creating environmentally friendly and sustainable processes. Oxygenases are enzymes capable of incorporating molecular oxygen (O2) into organic substrates with high regio- and stereoselectivities. By that, new functionalities can be incorporated into otherwise relatively inert organic molecules, yielding value-added products or synthons for synthetic routes.

This thesis provides an overview of the class of Rieske oxygenases (ROs). In addition to a literature review on ferredoxin-dependent enzymes with a focus on ROs, the thesis includes work on the three-component RO cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01. In nature, CDO catalyzes the initial step in the microbial biodegradation of aromatic xenobiotics, yielding vicinal cis-dihydro diols. The present work evaluated the feasibility, efficiency, and robustness of a cell-free reaction platform for CDO. In addition to the evaluation of protein expression and purification procedures, optimized reaction conditions for asymmetric hydroxylation reactions are reported. Problems related to O2 uncoupling in the established in vitro system for CDO have focused our attention on finding solutions to overcome this obstacle. It was found that the implementation of non-native redox partners into the CDO system significantly reduced H2O2 formation due to O2 uncoupling and contributed to a robust reaction system. Finally, a novel fusion approach to simplify the CDO reaction system has been evaluated. By fusing the reductase to the ferredoxin component of CDO, an efficient and simplified reaction system was established. This study serves as a blueprint for further fusion strategies for ROs.