School of Life Sciences and Health Engineering, Jiangnan University, Wuxi, 214122, China.
School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Nat Commun. 2024 Jul 9;15(1):5737. doi: 10.1038/s41467-024-50141-2.
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 GkOYE in both MBH and reduction reactions. By blocking H and H transfer pathways, we engineer GkOYE.8, which loses its reduction ability but enhances its MBH activity. The structural basis of MBH reaction catalyzed by GkOYE.8 is obtained through mutation studies and kinetic simulations. Furthermore, enantiocomplementary mutants GkOYE.11 and GkOYE.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.
探索天然酶的多功能性为扩展其合成应用提供了一个有前途的策略，特别是在非天然环境中催化具有挑战性的反应。在这项研究中，我们探索了老黄酶（OYE）的多功能性，以促进 Morita-Baylis-Hillman 反应（MBH 反应），利用 MBH 反应和还原反应之间的底物相似性。使用质谱和光谱技术，我们证实了 GkOYE 在 MBH 和还原反应中的多功能性。通过阻断 H 和 H 转移途径，我们对 GkOYE.8 进行了工程改造，使其失去还原能力但增强了 MBH 活性。通过突变研究和动力学模拟获得了 GkOYE.8 催化 MBH 反应的结构基础。此外，通过定向进化获得了对映互补突变体 GkOYE.11 和 GkOYE.13，它们能够接受各种芳香醛和烯烃作为底物。这项研究表明，利用底物相似性来解锁酶的功能，从而实现新的天然反应的催化是有潜力的。
