Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in CtFDH increased 6.5-fold the K_M, indicating that substrate binding was weakened. A 6.5-fold increase of k_cat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in CmFDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of CtFDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in CmFDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide.

中文翻译：

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NAD +依赖性甲酸脱氢酶中活性位点突变对碳酸氢盐转化为甲酸的功能影响

研究了甲基假丝酵母（CmFDH）和嗜热绒毛毛球菌（CtFDH）甲酸脱氢酶（FDHs）的突变体将碳酸氢盐转化为甲酸。碳酸氢盐不是FDH中氢化物转移反应的主要底物。选择所选择的突变，以使酶活性可以保持在适当的水平。在CtFDH中，活性位点的Asn120Cys突变使甲酸的酶失活（氧化），但作为底物浓度的函数，增加了碳酸氢盐的比活（还原）。CtFDH中的Asn120Cys突变使K _M增加6.5倍，表明底物结合减弱。k _cat增加6.5倍补偿了较低的亲和力，表明产品的释放得到改善。CmFDH中的相应突变Asn119Cys使两种底物的酶失活。分子动力学模拟表明，突变后活性位点的大小随底物的不同而变化，并且在突变体CtFDH的Asn120Cys中，碳酸氢盐的反应性位置优于甲酸。对于碳酸氢盐，活性位点扩大到足以将两个碳酸氢盐分子放置在那里的程度。在CmFDH中将Asn119变为庞大的Tyr或His时，需要改变活性位点以适应底物。保留了甲酸的活性，但不保留碳酸氢盐的活性。这项研究表明，FDHs的活性位点可以被彻底地修饰，