Structural Evolution of Enzymes from Antarctic Psychrotrophs and Molecular Mechanism for Their Higher Activities at Low Temperatures

由冷适应微生物产生的酶通常在低温下表现出更高的催化效率和比它们的嗜中温对应物更低的热稳定性。本文报道了由南极嗜冷菌Alteromonas sp. AS-11分泌的类枯草杆菌蛋白酶碱性丝氨酸蛋白酶Apa 2，它不仅在低温下具有较高的蛋白水解活性，而且比嗜中温枯草杆菌蛋白酶BPN '具有更高的热稳定性。Apa 2相对于枯草杆菌蛋白酶BPN'的比蛋白水解活性随着温度的降低而增加，尽管两种酶的最适温度是相同的。在0℃下，Apa 2的活性是枯草杆菌蛋白酶BPN '的9.9倍。它在57℃的半衰期比枯草杆菌蛋白酶BPN'长10倍，热变性的活化焓和熵也更高。Apa 2的这些特征支持这样的想法，即高催化效率所需的构象灵活性的结构决定因素可以与热不稳定性的结构决定因素分离。基因apa 2编码一个629个氨基酸的前原肽，含有一个283个残基的成熟酶，与枯草杆菌蛋白酶样丝氨酸蛋白酶具有显着的同源性。Apa 2的结构模型表明，二硫键、更多的盐桥和Ca^<2+>结合有助于其热稳定性，而其冷适应可能归因于环和β-转角或弯曲区域的结构柔性增加：

Enzymes produced by cold-adapted microorganisms generally exhibit higher catalytic efficiency at low temperatures and lower thermostability than their mesophilic counterparts. We here report on the subtilisin-like alkaline serine protease Apa2 secreted by Antarctic psychrotroph Alteromonas sp. AS-11, which is characterized by not only a higher proteolytic activity at low temperatures but also a greater thermostability than the mesophilic subtilisin BPN'. The specific proteolytic activity of Apa2 relative to subtilisin BPN' increased with decreasing temperature, although the optimal temperatures of both enzymes were the same. At 0℃, Apa2 was 9.9-fold more active than subtilisin BPN'. It had 10 times longer half-life time at 57℃ than subtilisin BPN' with higher activation enthalpy and entropy for thermal denaturation. These features of Apa2 support the idea that structural determinants , of conformational flexibility required for high catalytic efficiency can be separated from those of thermal instability. The gene apa2 encodes a 629-amino-acid pre-pro-peptide containing a mature enzyme of 283 residues that has significant homologies with subtilisin-like serine proteases. The structural model of Apa2 suggests that the disulfide bonds, a higher number of salt bridges, and Ca^<2+>binding contribute to its thermostability, while its cold-adaptation may be attributed to the increase in structural flexibility at loop and β-turn or bend regions: