Abstract
Exploring the promiscuity inherent in native enzymes presents a promising strategy for expanding their synthetic applications, particularly for catalyzing challenging reactions in non-native contexts. While considerable strides have been made in harnessing native enzymes for C-C bonding, certain reactions, such as the Morita-Baylis-Hillman reaction (MBH reaction), remain elusive to these biocatalysts. In this study, we explored the promiscuous potential of old yellow enzymes (OYEs) to facilitate the MBH reaction, leveraging substrate similarities between MBH reaction and the native reduction reaction. Initially, we substantiated the promiscuous nature of GkOYE in both MBH and reduction reactions through thorough analysis employing mass spectrometry and spectroscopic techniques. Following that, we manipulated functional selectivity by obstructing H− and H+ transfer pathways. Mutant GkOYE.8 eliminated the native reduction function while enhancing the MBH adduct yield by 141.4%, surpassing the wild type. Further insights into the structural basis of GkOYE.8 catalyzed MBH reaction were obtained through mutation studies and kinetic simulations, identifying C26 and E59 as key residues in the active center. To enhance the catalytic efficiency and stereoselectivity of GkOYE.8, we rationally designed mutants GkOYE.11 and GkOYE.13, which efficiently catalyzed the formation of MBH adducts, yielding (R)-3 (77.8% yield and 89:11 e.r.) and (S)-3 (63.1% yield and 23: 77 e.r.), respectively. Furthermore, it was found that the mutants GkOYE.11 and GkOYE.13 all showed catalytic activity against five p-substituent aldehydes and six-membered unsaturated alkenes. And the yield exhibited a gradual decrease as the electron-withdrawing strength of the p-substituents decreased. This study emphasizes the potential of leveraging substrate similarities across diverse reaction types to reveal new enzyme functionalities, enabling the catalysis of new-to-nature reactions.