Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
Department of Molecular and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia.
Appl Environ Microbiol. 2020 Aug 18;86(17). doi: 10.1128/AEM.00971-20.
Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon , which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by 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 Ca ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca-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. Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon , is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.
佩尔尼斯蛋白酶是一种枯草杆菌蛋白酶样蛋白酶，最初在生活在极端海洋环境中的嗜热古菌中被发现。由于经历了高温条件，佩尔尼斯蛋白酶表现出了非凡的稳定性和活性。佩尔尼斯蛋白酶在工业上具有重要的应用价值，因为它是少数几种具有朊病毒降解活性的蛋白酶之一。与其他细胞外枯草杆菌蛋白酶一样，佩尔尼斯蛋白酶以其无活性的前体形式（原佩尔尼斯蛋白酶）合成，需要经过成熟过程才能具有蛋白水解活性。中温菌枯草杆菌蛋白酶的成熟过程已经得到了详细的研究；然而，对于它们的嗜热同源物，如佩尔尼斯蛋白酶，人们对其成熟过程的了解较少。在这里，我们表明，在没有钙离子的情况下，原佩尔尼斯蛋白酶的结构是无定形的。与中温菌枯草杆菌蛋白酶不同，原佩尔尼斯蛋白酶需要钙离子才能采用适合其后续成熟的构象。除了从耐热 Tk-枯草杆菌蛋白酶中保守下来的几个钙离子结合位点外，佩尔尼斯蛋白酶还有一个带有钙离子结合模体的额外插入序列。我们证明了该插入序列对于佩尔尼斯蛋白酶在成熟过程中的有效折叠和稳定化的重要性。此外，对佩尔尼斯蛋白酶原肽的分析解释了原佩尔尼斯蛋白酶成熟对高温的需求。值得注意的是，原肽在高温下对佩尔尼斯蛋白酶催化结构域的抑制作用更强。解离后，只有在高温下原肽才会失稳，导致其降解，最终激活佩尔尼斯蛋白酶。我们的数据为理解耐热枯草杆菌蛋白酶自动激活机制提供了新的见解。来自嗜热生物的酶因其出色的稳定性和活性，在工业应用中尤为重要。佩尔尼斯蛋白酶来自嗜热古菌，是一种能够降解感染性朊病毒蛋白的蛋白酶，因此在消毒朊病毒污染的表面方面具有潜在的应用价值。与其他枯草杆菌蛋白酶类似，佩尔尼斯蛋白酶需要通过自动催化过程成熟成为活性蛋白酶。在本研究中，我们研究了佩尔尼斯蛋白酶的成熟过程，并表明该过程在高温下通过原肽特异性地受到调节。此外，我们证明了一个独特的钙离子结合插入对于成熟佩尔尼斯蛋白酶的稳定性的重要性。我们的研究结果提供了对耐热枯草杆菌蛋白酶自动激活的新认识，这可能会促进这些酶在商业用途上的发展。
