Laboratory of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, P. R. China.
Dalian Key Laboratory of Energy Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, P. R. China.
Chemistry. 2020 Dec 15;26(70):16611-16615. doi: 10.1002/chem.202003102. Epub 2020 Nov 16.
Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD-pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D-lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D-lactate at the expense of formate.
甲酸脱氢酶（FDH）已被广泛用于还原型烟酰胺腺嘌呤二核苷酸（NADH）的再生。为了利用烟酰胺胞苷二核苷酸（NCD）作为非天然氧化还原辅酶，需要有效地再生 NCDH，这仍然是一个挑战，因为 NCDH 是 NCD 的还原形式。在这里，我们展示了成功的 FDH 工程化以用于 NCDH 的再生。在 Pseudomonas sp. 101 （pseFDH）的 FDH 结构信息和 NAD-pseFDH 复合物的指导下，应用半理性策略设计突变文库并筛选与 NCD 相关的活性。最活跃的突变体达到了辅酶偏好从 NAD 到 NCD 的 3700 倍的转换。同源建模分析表明，这些突变体的辅酶结合口袋更小，并具有专门的疏水性相互作用，可用于 NCD。通过为依赖 NCD 的 D-乳酸脱氢酶提供动力，实现了 NCDH 的有效再生，该酶以甲酸为代价，通过计量和立体特异性还原丙酮酸来实现 D-乳酸的生成。
