Unlocking the function promiscuity of old yellow enzyme to catalyze asymmetric Morita-Baylis-Hillman reaction

Exploring the promiscuity of native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. In this study, we explore the promiscuous potential of old yellow enzymes (OYEs) to facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction reaction. Using mass spectrometry and spectroscopic techniques, we confirm promiscuity of Gk OYE in both MBH and reduction reactions. By blocking H - and H + transfer pathways, we engineer Gk OYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by Gk OYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants Gk OYE.11 and Gk OYE.13 are obtained by directed evolution, exhibiting the ability to accept various aromatic aldehydes and alkenes as substrates. This study demonstrates the potential of leveraging substrate similarities to unlock enzyme functionalities, enabling the catalysis of new-to-nature reactions. Exploring the promiscuity of native enzymes is a promising strategy for expanding their synthetic applications. Here, the authors show that old yellow enzymes (OYEs) can facilitate the Morita-Baylis-Hillman reaction (MBH reaction), leveraging substrate similarities between MBH reaction and reduction, and engineer GkOYE.8 with no reduction activity, but enhanced MBH activity.