Abstract

NAD⁺-dependent formate dehydrogenases (FDHs, E.C 1.2.1.2) catalyse the reversible reaction of CO₂ to formate ion (HCOO⁻) and reduces NAD⁺ molecule to NADH. Previously described FDHs from Chaetomium thermophilum (CtFDH) and Ceriporiopsis subvermispora (CsFDH) are active against formate and HCO₃^–. In this study, we examined the functional effects of active site mutations in Ct and Cs NAD⁺-dependent FDHs. The residues Ile94, Asn120, Val310, His312 at CtFDH and Asn312, Val313, Val331 at CsFDH are located in the active site. The effects of amino acid changes on catalytic properties and thermal stability of CtFDH and CsFDH revealed some interesting results compared with structurally equivalent positions that have been studied in the literature. The strongest effect was observed in CsFDH Val313Pro against HCO₃^–. The K_M value of the CsFDH Val313 enzyme for HCO₃^– substrate dramatically decreased, and enzyme activity increased. In CtFDH mutants, all enzymes except Val310Asn showed an increased k_cat value when K_M values increase. Analyses of results of mutant CtFDHs and CsFDHs give some promising results for CO₂ reduction as compared to the literature. Structural analyses of the substrate-binding site were done by homology modelling.

摘要

NAD ⁺依赖性甲酸脱氢酶（FDHs，EC 1.2.1.2）催化CO ₂可逆反应生成甲酸离子（HCOO ^-）并将NAD ⁺分子还原为NADH。先前描述的来自嗜热毛壳菌( Ct FDH) 和Ceriporiopsis subvermispora ( Cs FDH) 的 FDH 对甲酸盐和 HCO ₃ ^–具有活性。在这项研究中，我们检查了Ct和Cs NAD ⁺依赖性 FDH中活性位点突变的功能影响。残基 Ile94、Asn120、Val310、His312 在Ct FDH 和 Asn312、Val313、Val331 在Cs FDH 位于活性部位。与文献中研究的结构等效位置相比，氨基酸变化对Ct FDH 和Cs FDH 的催化性能和热稳定性的影响揭示了一些有趣的结果。在Cs FDH Val313Pro 中观察到对 HCO ₃ ^–的最强作用。Cs FDH Val313 酶对 HCO ₃ ^-底物的 K _M值显着降低，酶活性增加。在的Ct FDH突变体，不同之处Val310Asn所有酶显示增加ķ_猫值当K_中号值增加。与文献相比，突变体Ct FDH 和Cs FDH的结果分析给出了一些有希望的 CO ₂还原结果。底物结合位点的结构分析通过同源建模进行。