Department of Molecular Enzymology, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany.
Int J Mol Sci. 2023 Nov 8;24(22):16067. doi: 10.3390/ijms242216067.
Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from for its specificity toward NAD vs. NADP reduction. Starting from the NAD-specific wild-type FDH, key residues were exchanged to enable NADP binding on the basis of the NAD-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys) in the β-subunit of the enzyme is essential for the binding of NAD. RcFDH variants that had Glu exchanged for either a positively charged or uncharged amino acid had additional activity with NADP. The FdsB and FdsB variants also showed activity with NADP. Kinetic parameters for all the variants were determined and tested for activity in CO reduction. The variants were able to reduce CO using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.
甲酸盐脱氢酶催化甲酸盐向二氧化碳的可逆氧化。这些酶在 CO 还原中起着重要作用，并且是烟酰胺辅酶再生酶。最近，甲酸盐脱氢酶（尤其是含金属的甲酸盐脱氢酶）的 CO 还原活性已被进一步探索，以用于高效大气 CO 捕获。在这里，我们研究了 中的甲酸盐脱氢酶的烟酰胺结合位点，以了解其对 NAD 与 NADP 还原的特异性。从 NAD 特异性野生型 FDH 开始，根据 NAD 结合的冷冻电镜结构（PDB-ID：6TG9），交换关键残基以实现 NADP 结合。已经观察到，酶的β亚基中位置 157（赖氨酸）的赖氨酸对于 NAD 的结合是必需的。用带正电荷或不带电荷的氨基酸替换 Glu 的 RcFDH 变体具有与 NADP 结合的额外活性。FdsB 和 FdsB 变体也显示出与 NADP 的活性。所有变体的动力学参数均已确定，并在 CO 还原中进行了活性测试。该变体能够在与亚磷酸脱氢酶（PTDH）偶联的测定中使用 NADPH 作为电子供体还原 CO，该酶可将 NADPH 再生。这使得该酶适用于与其他使用 NADPH 的酶偶联的应用。
