Enantioselective Synthesis of Bicyclic Lactone Enabled by Tunnel Reshaping of Polycyclic Ketone Monooxygenase.

The Baeyer-Villiger monooxygenase (BVMO)-mediated oxidation of racemic cyclobutanone offers an attractive route to (1S,5R)-bicyclic lactone for the synthesis of unsaturated fatty acid prostaglandin. However, limited enantioselectivity restricts the applicability of this method. In this work, a thermostable BVMO PockeMO was engineered in the active site and substrate tunnel to obtain the quadruple mutant MU4 (N541V/N625W/I601Y/L226W). This mutant increased the ratio between the desired normal lactone (NL) and abnormal lactone (AL) from 1.1 to 43 (NL/AL ratio) compared to the wild type. Structural analysis revealed that the tunnel was reshaped and the N625W/I601Y mutation generated a gate-like structure to regulate the selectivity toward the racemic bicyclic ketone substrate. Notably, mutation N541V exerted a long-range epistatic effect on the tunnel mutations to modulate selectivity. Under a 200 mL scale, MU4 achieved an NL:AL ratio of 23 with 100 mM substrate. This study could enable the selective synthesis of chiral cyclolactone-containing natural products.