Lomonosov Moscow State University, Faculty of Chemistry, 119991, Moscow, Russian Federation.
Lomonosov Moscow State University, Faculty of Chemistry, 119991, Moscow, Russian Federation; Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, 119071, Moscow, Russian Federation.
Biochimie. 2024 Jan;216:194-204. doi: 10.1016/j.biochi.2023.10.016. Epub 2023 Nov 2.
NAD-dependent formate dehydrogenase (FDH, EC 1.2.1.2) from the bacterium Staphylococcus aureus (SauFDH) plays an important role in the vital activity of this bacterium, especially in the form of biofilms. Understanding its mechanism and structure-function relationship can help to find special inhibitors of this enzyme, which can be used as medicines against staphylococci. The gene encoding SauFDH was successfully cloned and expressed in our laboratory. This enzyme has the highest k value among the described FDHs and also has a high temperature stability compared to other enzymes of this group. That is why it can also be considered as a promising catalyst for NAD(P)H regeneration in the processes of chiral synthesis with oxidoreductases. In this work, the principle of rational design was used to improve SauFDH catalytic efficiency. After bioinformatics analysis of the amino acid sequence in combination with visualization of the enzyme structure (PDB 6TTB), 9 probable catalytically significant positions 119, 194, 196, 217-219, 246, 303 and 323 were identified, and 16 new mutant forms of SauFDH were obtained and characterized by kinetic experiments. The introduction of the mentioned substitutions in most cases leads to a decrease in stability at high temperatures and an increase at low temperatures. Substitutions in positions 119 and 194 lead to a decreasing of K. A consistent decrease in the Michaelis constant in the Ile-Val-Ala-Gly series at position 119 of SauFDH is shown. K of mutant SauFDH V119G decreased by 27 times compared to the wild-type enzyme. After substitution Phe194Val K decreased by 3.5 times. The catalytic constant for this mutant form practically did not change. For this mutant form, an increase in catalytic efficiency was demonstrated through the use of a multicomponent buffer system.
来自金黄色葡萄球菌（SauFDH）的烟酰胺腺嘌呤二核苷酸（NAD）依赖性甲酸脱氢酶（FDH，EC 1.2.1.2）在该细菌的生命活动中起着重要作用，特别是在生物膜形式中。了解其机制和结构-功能关系有助于找到该酶的特殊抑制剂，可将其用作抗葡萄球菌的药物。我们实验室成功地克隆和表达了编码 SauFDH 的基因。与该组的其他酶相比，该酶具有描述的 FDH 中最高的 k 值，并且还具有较高的温度稳定性。这就是为什么它也可以被认为是手性合成中氧化还原酶 NAD（P）H 再生的有前途的催化剂。在这项工作中，使用合理设计的原理来提高 SauFDH 的催化效率。在结合酶结构可视化（PDB 6TTB）进行氨基酸序列的生物信息学分析后，鉴定了 9 个可能的催化关键位置 119、194、196、217-219、246、303 和 323，并获得了 16 种新的 SauFDH 突变体形式并通过动力学实验进行了表征。在大多数情况下，引入这些取代基会导致高温下稳定性降低，低温下稳定性增加。位置 119 和 194 的取代导致 K 值降低。在 SauFDH 位置 119 的 Ile-Val-Ala-Gly 系列中，米氏常数一致降低。与野生型酶相比，突变 SauFDH V119G 的 K 值降低了 27 倍。取代苯丙氨酸 194 缬氨酸后 K 值降低了 3.5 倍。该突变体形式的催化常数几乎没有变化。对于这种突变形式，通过使用多组分缓冲系统证明了催化效率的提高。
