An Optimized System for the Study of Rieske Oxygenase‐catalyzed Hydroxylation Reactions In vitro

Abstract

Rieske non-heme iron oxygenases (ROs) are primarily known for their ability to catalyze the stereoselective formation of vicinal cis-diols in a single step, endowing valuable products for pharmaceutical and chemical applications. In addition, ROs can catalyze several other oxidation reactions with high regio-and stereoselectivity and typically broad substrate scope. Owing to their dependence on multicomponent electron transfer, the majority of synthetic applications of ROs relies on recombinant whole-cell catalysts. In this context, important properties of the multicomponent system that determine the catalytic efficiency, including electron transfer via redox partner proteins, stability and uncoupling, have been investigated to a lesser extent in recent years. Here, we show for one of the most prominent ROs, the cumene dioxygenase from Pseudomonas fluorescens IP01 (CDO) that by developing and optimizing an efficient in vitro system, high catalytic activities can be achieved. In addition, we highlight that an efficient and continuous supplementation of electrons to the oxygenase is required to sustain their catalytic activity, while uncoupling can be a major limitation in CDO efficiency and stability.

Developing Hybrid Systems to Address O2Uncoupling in Multi-Component Rieske Oxygenases

“…Cell harvesting, washing and lysis were performed according to a previously optimized purification protocol. 25 After incubation of the clarified lysate with 2 mL Ni-sepharose resin (1 column volume (CV) corresponds to 2 mL) for 1 h and washing with 5 CV of washing buffer (50 mM SPB, 30 mM imidazole, 300 mM NaCl, 10 % glycerol, pH 7.2), elution was performed using defined amounts of elution buffer (50 mM SPB, 400 mM imidazole, 300 mM NaCl, 10 % glycerol, pH 7.2). For CDO protein components, elution fractions 0.5 to 2xCVs were collected for downstream sample preparation.…”

“…Final protein concentrations were determined using a colorimetric Coomassie protein assay as described previously. 25…”

“…The present study evaluates the impact of ROS formation due to O 2 uncoupling on cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a suitable model enzyme system representing a well-studied three-component RO. 23–25 Therefore, we chose an in vitro setup for CDO based on a balanced ratio of RO redox components and a NADH cofactor regeneration system using glucose dehydrogenase (GDH) to maintain cofactor supply. 25 According to our previous results, the formation of ROS driven solely by RO-Red of CDO (CDO-Red) was demonstrated, 25 reaching a concentration of more than 4 mM H 2 O 2 after 24 h in the presence of the NADH-cofactor regeneration system.…”

“…23–25 Therefore, we chose an in vitro setup for CDO based on a balanced ratio of RO redox components and a NADH cofactor regeneration system using glucose dehydrogenase (GDH) to maintain cofactor supply. 25 According to our previous results, the formation of ROS driven solely by RO-Red of CDO (CDO-Red) was demonstrated, 25 reaching a concentration of more than 4 mM H 2 O 2 after 24 h in the presence of the NADH-cofactor regeneration system. In addition, the use of catalase was found to be essential to maintain up to 40 % conversion of 10 mM indene ( 1 ) to 1H-indenol ( 1a ) and cis -indane diol ( 1b ) (Scheme 2).…”

“…Redox interactions between associated CDO components with PDR (or VanB, not shown) within a hybrid RO system, exemplified for the conversion of indene ( 1 ) into 1 H -indenol ( 1a ) and cis -1,2-indanediol ( 1b ) catalyzed by CDO in the presence of molecular oxygen and NADH. 25 …”