A novel mechanism of protein thermostability: a unique N-terminal domain confers heat resistance to Fe/Mn-SODs.

Superoxide dismutases (SODs), especially thermostable SODs, are widely applied in medical treatments, cosmetics, food, agriculture, and other industries given their excellent antioxidant properties. A novel thermostable cambialistic SOD from Geobacillus thermodenitrificans NG80-2 exhibits maximum activity at 70°C and high thermostability over a broad range of temperatures (20–80°C). Unlike other reported SODs, this enzyme contains an extra repeat-containing N-terminal domain (NTD) of 244 residues adjacent to the conserved functional SODA domain. Deletion of the NTD dramatically decreased its optimum active temperature (OAT) to 30°C and also impaired its thermostability. Conversely, appending the NTD to a mesophilic counterpart from Bacillus subtilis led to a moderately thermophilic enzyme (OAT changed from 30 to 55°C) with improved heat resistance. Temperature-dependant circular dichroism analysis revealed the enhanced conformational stability of SODs fused with this NTD. Furthermore, the NTD also contributes to the stress resistance of host proteins without altering their metal ion specificity or oligomerisation form except for a slight effect on their pH profile. We therefore demonstrate that the NTD confers outstanding thermostability to the host protein. To our knowledge, this is the first discovery of a peptide capable of remarkably improving protein thermostability and provides a novel strategy for bioengineering thermostable SODs.

超氧化物歧化酶（SOD），尤其是热稳定的SOD，由于其优异的抗氧化特性，广泛应用于医疗、化妆品、食品、农业和其他行业。来自嗜热脱氮地芽孢杆菌NG80 - 2的一种新型热稳定兼性SOD在70°C时表现出最大活性，并且在较宽的温度范围（20 - 80°C）内具有高热稳定性。与其他已报道的SOD不同，这种酶包含一个额外的含重复序列的N末端结构域（NTD），该结构域由244个氨基酸残基组成，与保守的功能性SODA结构域相邻。NTD的缺失使其最适活性温度（OAT）大幅降低至30°C，并且也损害了其热稳定性。相反，将NTD添加到来自枯草芽孢杆菌的中温对应物上，产生了一种适度嗜热的酶（OAT从30°C变为55°C），其耐热性得到提高。温度依赖性圆二色性分析显示，与该NTD融合的SOD构象稳定性增强。此外，NTD还有助于宿主蛋白的抗逆性，除了对其pH曲线有轻微影响外，不会改变其金属离子特异性或寡聚形式。因此，我们证明NTD赋予宿主蛋白出色的热稳定性。据我们所知，这是首次发现一种能够显著提高蛋白质热稳定性的肽，并为生物工程改造热稳定SOD提供了一种新策略。