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

由冷适应微生物和鱼产生的酶通常在低温下表现出更高的催化效率，而热稳定性比它们的中温对应物低。来自南极嗜冷菌希瓦氏菌AS-11的嗜冷碱性丝氨酸蛋白酶Apa 1由枯草杆菌蛋白酶样区域（293个残基）和插入枯草杆菌蛋白酶β8和β9链之间的附加区域（148个残基）组成。Apa 1的晶体结构已在1.8 nm分辨率下得到解析和精细化。枯草杆菌蛋白酶样结构域的结构与枯草杆菌蛋白酶的结构相似，但插入结构域有一个新的折叠，包含11个β链和2个α螺旋，主链原子的B因子分析表明插入结构域具有更高的柔性，并且在不对称单元中的两个Apa 1分子的比较表明插入结构域相对于枯草杆菌蛋白酶具有旋转。Apa 1结构与嗜温同源酶模型的比较表明，Apa 1失去了一个盐B 关于我们 在嗜温对应物中观察到的枯草杆菌蛋白酶样结构域和插入结构域之间的脊。这些结果表明，插入物的柔性有助于Apa 1在低温下的催化效率。这已经通过嗜温枯草杆菌蛋白酶Carlsberg的突变分析得到证实。本文比较了来自希瓦氏菌（Shewanella sp. AS-11，SAK）和嗜温菌（mesophilie E. coli（EAK）。SAK的特点是向低温的最佳活性的偏移和较低的热稳定性相比，EAK。在10℃时，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。SAK的模拟结构显示，与EAK相比，盐桥和阳离子-π相互作用的数量减少。这些结果表明，SAK是一个更灵活的结构冷适应。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