Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.