A Thermostable Heme Protein Fold Adapted for Stereoselective C−H Bond Hydroxylation Using Peroxygenase Activity
Tuhin Das, Eva F. Hayball, Alix C. Harlington, Stephen G. Bell
Abstract
Abstract Thermostable protein folds of natural and synthetic origin are highly sought‐after templates for biocatalyst generation due to their enhanced stability to elevated temperatures which overcomes one of the major limitations of applying enzymes for synthesis. Cytochrome P450 enzymes (CYPs) are a family of heme‐thiolate monooxygenases that catalyse the oxidation of their substrates in a highly stereo‐ and regio‐selective manner. The CYP enzyme (CYP107PQ1) from the thermophilic bacterium Meiothermus ruber binds the norisoprenoid β‐ionone and was employed as a scaffold for catalyst design. The I‐helix was modified to convert this enzyme from a monooxygenase into a peroxygenase (CYP107PQ1QE), enabling the enantioselective oxidation of β‐ionone to ( S )‐4‐hydroxy‐β‐ionone (94 % e.e.). The enzyme was resistant to 20 mM H 2 O 2 , 20 % (v/v) of organic solvent, supported over 1700 turnovers and was fully functional after incubation at 60 °C for 1 h and 30 °C for 365 days. The reaction was scaled‐up to generate multi milligram quantities of the product for characterisation. Overall, we demonstrate that sourcing a CYP protein fold from an extremophile enabled the design of a highly stable enzyme for stereoselective C−H bond activation only using H 2 O 2 as the oxidant, providing a viable strategy for future biocatalyst design.