Tuhin Das, Eva F. Hayball, Alix C. Harlington, Stephen Graham Bell
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引用次数: 0
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 H2O2, 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 H2O2 as the oxidant, providing a viable strategy for future biocatalyst design.
期刊介绍:
ChemBioChem (Impact Factor 2018: 2.641) publishes important breakthroughs across all areas at the interface of chemistry and biology, including the fields of chemical biology, bioorganic chemistry, bioinorganic chemistry, synthetic biology, biocatalysis, bionanotechnology, and biomaterials. It is published on behalf of Chemistry Europe, an association of 16 European chemical societies, and supported by the Asian Chemical Editorial Society (ACES).