Enzymatic biocatalysis can be limited by the necessity of soluble cofactors. Here, we introduced PEGylated nicotinamide adenine dinucleotide (NAD(H)) swing arms to two covalently fused dehydrogenase enzymes to eliminate their nicotinamide cofactor requirements. A formate dehydrogenase and cytosolic malate dehydrogenase were connected via SpyCatcher-SpyTag fusions. Bifunctionalized polyethylene glycol chains tethered NAD(H) to the fusion protein. This produced a formate:malate oxidoreductase that exhibited cofactor-independent ping-pong kinetics with predictable Michaelis constants. Kinetic modeling was used to explore the effective cofactor concentrations available for electron transfer in the complexes. This approach could be used to create additional cofactor-independent transhydrogenase biocatalysts by swapping fused dehydrogenases.

中文翻译：

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通过将脱氢酶与聚乙二醇化的辅因子摆臂融合来形成甲酸盐：苹果酸氧化还原酶。

酶促生物催化作用可能受可溶性辅因子的限制。在这里，我们向两个共价融合的脱氢酶引入了聚乙二醇化烟酰胺腺嘌呤二核苷酸（NAD（H））摆臂，以消除它们对烟酰胺辅因子的需求。甲酸酯脱氢酶和胞质苹果酸脱氢酶通过SpyCatcher-SpyTag融合体连接。双功能聚乙二醇链将NAD（H）拴在融合蛋白上。这产生了甲酸酯：苹果酸氧化还原酶，其表现出具有可预测的米氏常数的独立于辅因子的乒乓动力学。动力学建模用于探索可用于电子在复合物中转移的有效辅因子浓度。该方法可用于通过交换融合的脱氢酶来创建其他不依赖辅因子的转氢酶生物催化剂。