Structural evolution and molecular mechanism of cold-active enzymes from Antarctic psychrophiles

冷适应微生物和鱼类产生的酶在低温下通常表现出更高的催化效率，而热稳定性较低。来自南极嗜冷菌Shewanella sp. AS-11的嗜冷碱性丝氨酸蛋白酶Apa1由一个类似枯草菌素的区域（293个残基）和一个插入在枯草菌素β8和β9链之间的附加区域（148个残基）组成。Apa1的晶体结构已在1.8Å分辨率下得到求解和细化。类枯草菌素结构域与枯草菌素结构域相似，但插入结构域有一个包含11条β链和2条α螺旋的新折叠。对主链原子的b因子分析表明，插入物具有更高的柔韧性，对不对称单元中两个Apa1分子的比较表明，插入物相对于枯草菌素样结构域有旋转。Apa1的结构与中温同源酶模型的比较表明，Apa1在枯草杆菌样结构域和插入结构域之间损失了一个盐区。这些结果表明，插入物的柔韧性有助于Apa1在低温下的催化效率。这已被嘉士伯中温枯草杆菌的突变分析证实。嘉士伯枯草杆菌β8链和β9链β-转位处Pro209向Gly突变导致酶活性升高。比较了希瓦氏菌AS-11 （SAK）和大肠杆菌（EAK）两种乙酸激酶的结构和功能特性。与EAK相比，SAK的最佳活性向低温转移，热稳定性较低。在10℃条件下，SAK的催化效率（Kcat/Km）是EAK的12倍。SAK催化的两个反应方向的活化焓和熵均低于EAK。与EAK相比，SAK的模型结构显示出盐桥和阳离子- π相互作用的数量减少。这些结果表明，SAK具有较强的冷适应性，结构更灵活。少

Enzymes produced by cold-adapted microorganisms and fish generally exhibit higher catalytic efficiency at low temperatures and lower thermostability than their mesophilic counterparts. The psychrophilic alkaline serine protease Apa1 from the Antarctic psychrotroph Shewanella sp. AS-11 consists of a subtilisin-like region (293 residues) and an additional region (148 residues) that is inserted between β8 and β9 strands of subtilisins. The crystal structure of Apa1 has been solved and refined at 1.8Å resolution. The structure of subtilisin-like domain is similar to those of subtilisins, but the insert domain has a new fold containing 11β strands and 2 α helices.Analysis of B-factors for main chain atoms reveals a higher flexibility of the insert and the comparison of two Apa1 molecules in the asymmetric unit demonstrates a rotation of the insert relative to the subtilisin-like domain.Comparison of Apa1 structure with the model of mesophilie homologous enzyme shows that Apa1 loses a salt b … More ridge between subtilisin-like and insert domains observed in the mesophilic counterpart. These results suggest that the flexibility of the insert contributes to the catalytic efficiency of Apa1 at low temperatures. This has been confirmed by mutational analyses of mesophilic subtilisin Carlsberg. The mutation of Pro209 to Gly at β-turn between β8 and β9 strands of subtilisin Carlsberg caused the increase in enzymatic activity.The structural and functional properties were compared between two acetate kinases from Shewanella sp. AS-11 (SAK) and mesophilie E. coli (EAK). SAK was characterized by a shift of the optimum activity towards low temperatures and by a lower thermal stability compared with EAK. The catalytic efficiency (Kcat/Km) of SAK was 12-fold higher than that of EAK at 10℃. The activation enthalpy and entropy in both reaction directions catalyzed by SAK were lower than those by EAK, respectively. The modeled structure of SAK shows the reduced numbers of salt bridges and cation-pi interactions compared with EAK. These results indicate that SAK is cold-adapted with a more flexible structure. Less