Ma Wei, Geng Qiang, Chen Cheng, Zheng Yu-Cong, Yu Hui-Lei, Xu Jian-He
State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Meilong Road 130, Shanghai, 200237, China.
Chembiochem. 2023 Oct 17;24(20):e202300390. doi: 10.1002/cbic.202300390. Epub 2023 Aug 28.
Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (k /K ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.
烟酰胺腺嘌呤二核苷酸（NADH）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）是氧化/还原生物转化的主要氢供体。与甲酸脱氢酶（FDHs）偶联的NAD(P)H再生系统是一种理想的方法。然而，大多数天然FDHs依赖于NAD，与其他酶工具（如葡萄糖脱氢酶）相比，其反应活性不足。一种高效且有竞争力的利用NADP的FDH需要NADPH再生系统的可用性和稳健性。在此，我们报道了来自都柏林念珠菌的一种新型FDH（CdFDH）的工程改造，通过结构导向的理性/半理性设计，它没有严格的NAD偏好。一种组合突变体CdFDH-M4（D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R）的催化效率（k /K ）提高了75倍。此外，CdFDH-M4已成功应用于多种不对称氧化/还原过程，辅因子的总周转数（TTNs）在135至986之间，这使其在需要NADPH的生物催化转化中具有潜在应用价值。
