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

南极嗜冷菌冷活性酶的结构演化及分子机制

基本信息

批准号：
15380074
负责人：
WATANABE Keiichi
金额：
$ 10.3万
依托单位：
Saga University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

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

由冷适应微生物和鱼类产生的酶在低温下通常表现出比它们的中温对应物更高的催化效率和更低的热稳定性。南极嗜冷菌Shewanella sp.的嗜冷碱性丝氨酸蛋白酶APA1。AS-11由一个枯草杆菌蛋白样区(293个残基)和一个插入在β8和β9链之间的附加区(148个残基)组成。在1.8Å分辨率下，对APA1的晶体结构进行了求解和精化。类枯草杆菌蛋白结构域的结构与枯草杆菌蛋白相似，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因子分析表明插入片段具有较高的灵活性，不对称单元中的两个apA1分子的比较表明插入片段相对于类枯草杆菌蛋白结构域有旋转。与中亲同源酶模型的比较表明，apa1失去了一个盐b…在中温对应物中观察到更多的枯草杆菌素样结构域和插入结构域之间的隆起。这些结果表明，插入物的柔性有助于APA1在低温下的催化效率。这一点已被嘉士伯中温枯草杆菌的突变分析所证实。枯草杆菌β8链和β9链之间Pro209突变为Gly导致了酶活性的增加。AS-11(SAK)和嗜中性菌(EAK)。与EAK相比，SAK的最适活性向低温方向移动，热稳定性较低。在10℃时，SAK的催化效率(Kcat/Km)是EAK的12倍。SAK在两个反应方向上的活化热和活化熵均低于EAK。SAK的模拟结构显示，与EAK相比，盐桥和阳离子-pi相互作用的数量减少。这些结果表明SAK是冷适应的，具有更灵活的结构。较少