Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.
Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.
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DOI：
10.1006/jmbi.1996.0797
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发表时间：
1997-03
影响因子：
5.6
通讯作者：
Gerlind Wallon;G. Kryger;Susan T. Lovett;Tairo Oshima;Dagmar Ringe;G. Petsko
中科院分区：
文献类型：
--
作者：
Gerlind Wallon;G. Kryger;Susan T. Lovett;Tairo Oshima;Dagmar Ringe;G. Petsko
蛋白质稳定性的基础已被研究的结构比较嗜热酶与他们的中温同行。已经发现许多特性可以有助于嗜热蛋白质的稳定，但是没有一个是唯一能够赋予热稳定性的。用分子置换法测定了嗜温菌大肠杆菌和鼠伤寒沙门氏菌3-异丙基苹果酸脱氢酶（IPMDH）的晶体结构。对E.大肠杆菌酶的分离度为2.1A，R因子为17.3%，S.鼠伤寒沙门氏菌酶在1.7 A分辨率下的R因子为19.8%。比较了这三种结构，阐明了T.嗜热酶设计了一种在嗜热酶的疏水核心中产生空腔的突变体，以研究堆积密度对热稳定性的重要性。对该突变体的结构进行了分析。嗜热酶的主要稳定特征是盐桥数量的增加、额外的氢键、成比例更大且更疏水性的亚基界面、缩短的N和C末端以及更多的脯氨酸残基。T.嗜热菌IPMDH导致32 A3的空腔，但未观察到对突变体的活性和热稳定性的显著影响。
The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.