Kurt Sinem, Ordu Emel
Department of Molecular Biology and Genetics, Faculty of Arts and Science, Yildiz Technical University, Istanbul, Turkey.
Department of Molecular Biology and Genetics, Faculty of Science, Gebze Technical University, Kocaeli, Turkey.
Appl Microbiol Biotechnol. 2021 Apr;105(7):2787-2798. doi: 10.1007/s00253-021-11232-y. Epub 2021 Mar 22.
NAD-dependent formate dehydrogenases (FDHs) are extensively used in the regeneration of NAD(P)H and the reduction of CO to formate. In addition to their industrial importance, FDHs also play a crucial role in the maintenance of a reducing environment to combat oxidative stress in plants. Therefore, it is important to investigate the response of NAD-dependent FDH against both temperature and HO, to understand the defense mechanisms, and to increase its stability under oxidative stress conditions. In the present study, we characterized the oxidative and thermal stability of NAD-dependent FDH isolated from cotton, Gossypium hirsutum (GhFDH), by investigating the effect of Met/Leu substitutions in the positions of 225, 234, and 243. Results showed that the single mutant, M234L (0.72 s mM), and the triple mutant, M225L/M234L/M243L (0.55 s mM), have higher catalytic efficiency than the native enzyme. Substitution of methionine by leucine on the position of 243 increased the free energy gain by 670 J mol. The most remarkable results in chemical stability were seen for double and triple mutants, cumulatively. Double and triple substitution of Met to Leu (M225L/M243L and M225L/M243L/M234L) reduce the k by a factor of 2 (12.3×10 and 12.8×10 s, respectively.Key points• The closer the residue to NAD, in which we substituted methionine to leucine, the lower the stability against HO we observed.• The significant gain in the T value for the M243L mutant was observed as +5°C.• Residue 234 occupies a critical position for oxidation defense mechanisms. Graphical abstract (a) Methionine amino acids on the protein surface are susceptible to oxidative stress and can be converted to methionine sulfoxide by reactive oxygen derivatives (such as hydrogen peroxide). Therefore, they are critical regions in the change of protein conformation and loss of activity. (b) Replacing the amino acid methionine, which is susceptible to oxidation due to the sulfur group, with the oxidation-resistant leucine amino acid is an important strategy in increasing oxidative stability.
依赖烟酰胺腺嘌呤二核苷酸（NAD）的甲酸脱氢酶（FDHs）被广泛用于NAD（P）H的再生以及将CO还原为甲酸。除了其工业重要性外，FDHs在维持植物对抗氧化应激的还原环境中也起着关键作用。因此，研究依赖NAD的FDH对温度和过氧化氢（HO）的响应，以了解其防御机制，并提高其在氧化应激条件下的稳定性非常重要。在本研究中，我们通过研究棉株陆地棉（GhFDH）中225、234和243位的甲硫氨酸/亮氨酸替换的影响，来表征依赖NAD的FDH的氧化稳定性和热稳定性。结果表明，单突变体M234L（0.72 s mM）和三突变体M225L/M234L/M243L（0.55 s mM）比天然酶具有更高的催化效率。在243位将甲硫氨酸替换为亮氨酸使自由能增加670 J/mol。在化学稳定性方面，双突变体和三突变体的结果最为显著。甲硫氨酸到亮氨酸的双替换和三替换（M225L/M243L和M225L/M243L/M234L）分别使k降低了2倍（分别为12.3×10和12.8×10 s）。要点：• 我们将甲硫氨酸替换为亮氨酸的残基距离NAD越近，观察到的对HO的稳定性越低。• M243L突变体的T值显著增加了5°C。• 残基234在氧化防御机制中占据关键位置。图形摘要：（a）蛋白质表面的甲硫氨酸氨基酸易受氧化应激影响，可被活性氧衍生物（如过氧化氢）转化为甲硫氨酸亚砜。因此，它们是蛋白质构象变化和活性丧失的关键区域。（b）用抗氧化的亮氨酸氨基酸替换因含硫基团而易氧化的甲硫氨酸氨基酸是提高氧化稳定性的重要策略。
