Engineered alcohol oxidases catalyse transesterification in aqueous media without competing hydrolysis

Abstract Transesterification reactions are fundamental transformations in organic chemistry. However, performing these reactions in aqueous media is challenging due to the competing hydrolysis reaction. Kinetic control and the use of activated acyl donors offer potential solutions, but they are difficult to implement. In this study, we present a mutant of alcohol oxidase from Phanerochaete chrysosporium ( Pc AOx-VPN) that exhibits no hydrolytic activity. Its hydrophobic active site efficiently excludes water, enabling irreversible transesterification reactions in water—previously considered impossible—thereby opening novel opportunities for chemical synthesis. Remarkably, Pc AOx-VPN, as a flavin-containing enzyme, catalyzes transesterification, a promiscuous activity not previously known in this enzyme class, expanding the functional repertoire of these versatile enzymes.