Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor
Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor
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用于非天然氧化还原辅因子再生的甲酸脱氢酶的结构引导设计
DOI：
10.1002/chem.202003102
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发表时间：
2020-11-16
影响因子：
4.3
通讯作者：
Zhao, Zongbao K.
中科院分区：
文献类型：
--
作者：
Guo, Xiaojia;Wang, Xueying;Zhao, Zongbao K.
甲酸脱氢酶（FDH）被广泛用于还原烟酰胺腺嘌呤二核苷酸（NADH）的再生。利用烟酰胺胞嘧啶二核苷酸（NCD）作为一种非天然氧化还原辅助因子，仍然具有挑战性，因为NCD的还原形式NCDH必须有效地再生。在这里，我们展示了FDH在NCDH再生中的成功工程。以假单胞菌101 （Pseudomonas sp. 101, pseFDH）和nadd -pseFDH复合物的结构信息为指导，采用半理性策略设计突变文库和筛选非传染性疾病连锁活性。最活跃的突变体达到了从NAD到NCD的辅助因子偏好切换3700倍。同源性建模分析表明，这些突变体具有减少的辅因子结合口袋和专门的疏水相互作用。NCDH的高效再生是通过驱动ncd依赖的d -乳酸脱氢酶来实现的，以化学计量和立体定向的方式将丙酮酸还原为d -乳酸，而牺牲甲酸。
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.