NAD+-dependent formate dehydrogenase from Staphylococcus aureus (SauFDH) is one of the key enzymes responsible for the survival of this pathogen in the form of biofilms. 3D structure of the enzyme might be helpful in the search for highly specific SauFDH inhibitors that can be used as antibacterial agents exactly against S. aureus biofilms. Here, we prepared a recombinant SauFDH in Escherichia coli cells with a yield of 1 g target protein per liter medium. The developed procedure for the enzyme purification allowed to obtain 400 mg of homogenous enzyme with 61% yield. The specific activity of the purified recombinant SauFDH was 20 U per mg protein, which was 2 times higher than the previously reported activities of formate dehydrogenases. We also found crystallization conditions in the course of two rounds of optimization and obtained 200- and 40-µm crystals for the SauFDH apo- and holoenzymes, respectively. X-ray analysis using synchrotron X-ray sources produced diffraction data sufficient for solving the three-dimensional structures of the apo- and holoenzymes with the resolution of 2.2 and 2.7 Å, respectively. Crystals of the apo- and holoforms of SauFDH had different crystal space groups, which suggest coenzyme binding in the SauFDH holoenzyme.

中文翻译：

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金黄色葡萄球菌高活性重组甲酸脱氢酶的制备和结晶

来自金黄色葡萄球菌 (SauFDH) 的 NAD+ 依赖性甲酸脱氢酶是负责该病原体以生物膜形式存活的关键酶之一。该酶的 3D 结构可能有助于寻找高度特异性的 SauFDH 抑制剂，这些抑制剂可用作完全对抗金黄色葡萄球菌生物膜的抗菌剂。在这里，我们在大肠杆菌细胞中制备了一种重组 SauFDH，每升培养基的产量为 1 g 目标蛋白。用于酶纯化的开发程序允许以 61% 的产率获得 400 mg 同质酶。纯化后的重组 SauFDH 比活性为 20 U/mg 蛋白质，比之前报道的甲酸脱氢酶活性高 2 倍。我们还在两轮优化过程中找到了结晶条件，分别为 SauFDH 载脂蛋白和全酶获得了 200 和 40 微米的晶体。使用同步加速器 X 射线源的 X 射线分析产生的衍射数据足以解析载脂蛋白和全酶的三维结构，分辨率分别为 2.2 和 2.7 Å。SauFDH 的载脂蛋白和全息型的晶体具有不同的晶体空间群，这表明 SauFDH 全酶中存在辅酶结合。