Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix. Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca²⁺ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca²⁺ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca²⁺-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca²⁺-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.

中文翻译：

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深入了解嗜热古生气单胞菌多年生菌中类似枯草蛋白酶的蛋白酶-鸟氨酸的成熟。

Pernisine是一种枯草杆菌蛋白酶样的蛋白酶，最初在生活在极端海洋环境中的超嗜热古细菌Aeropyrum pernix中发现。由于pernix遇到的高温条件，pernisine显示出出色的稳定性和活性。对于工业用途，Pernisine是令人感兴趣的，因为Pernisine是少数具有demonstrated病毒降解活性的蛋白酶之一。像其他细胞外枯草杆菌蛋白酶一样，pernisine是以其非活性形式（pro-pernisine）合成的，其需要经过成熟才能具有蛋白水解活性。中温枯草杆菌蛋白酶的成熟过程已被详细研究。然而，关于它们的嗜热同系物如鸟氨酸的成熟知之甚少。在这里，我们表明，在没有Ca ²⁺离子的情况下，前pernisine的结构是无序的。与中温枯草杆菌蛋白酶不同，前pernisine需要Ca ²⁺离子采用适合其后续成熟的构象。除了几种Ca ²⁺从热稳定的Tk-枯草杆菌蛋白酶中保守的结合位点，pernisine具有带有Ca ²⁺结合基序的附加插入序列。我们证明了此插入物对pernisine成熟期间有效折叠和稳定化的重要性。此外，对pernisine前肽的分析解释了per-inesine成熟所需的高温。值得注意的是，前肽在高温下更有效地抑制了鸟氨酸的催化结构域。解离后，原肽仅在高温下不稳定，这导致其降解并最终导致鸟氨酸激活。我们的数据提供了对热稳定的枯草杆菌蛋白酶自动激活机制的新见解。