Mutation at a single acidic amino acid enhances the halophilic behaviour of malate dehydrogenase from Haloarcula marismortui in physiological salts.
Mutation at a single acidic amino acid enhances the halophilic behaviour of malate dehydrogenase from Haloarcula marismortui in physiological salts.
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单个酸性氨基酸的突变增强了 Haloarcula marismortui 在生理盐中的苹果酸脱氢酶的嗜盐行为。
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
Giuseppe Zaccai
中科院分区：
文献类型：
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作者：
D. Madern;Claude Pfister;Giuseppe Zaccai
在26嗜盐蛋白的氨基酸组成的统计分析中，24显示酸性氨基酸的增加和碱性氨基酸的减少相比，它们的非嗜盐同源物。采用定点突变的方法，研究了海洋盐腔菌苹果酸脱氢酶（MalDH）的酸性残基在嗜盐适应中的作用。在所有40种非嗜盐同源蛋白中，与嗜盐MalDH中的E243对齐的位置被非酸性氨基酸占据，最常见的是精氨酸。构建了嗜盐性MalDH的E243 R突变体，在大肠杆菌中高效表达，复性并纯化。与野生型酶相比，其盐依赖性催化活性不受影响，并且两种蛋白质对其底物具有相同的Km值。在不同的生理盐（NaCl或KCl）和温度条件下的突变体的变性的抗性进行了比较，野生型蛋白质和经典的准热力学参数的解释。突变体比野生型蛋白更嗜盐;它对温度更敏感，并且需要显著更高浓度的NaCl或KCl以获得等同的稳定性。这些结果突出了酸性氨基酸在嗜盐行为中的作用，并与这些氨基酸协同作用以组织水合离子结合蛋白质的模型一致。
In a statistical analysis of the amino acid compositions of 26 halophilic proteins, 24 showed an increase in acidic amino acids and a decrease in basic ones when compared to their non-halophilic homologues. The role of acidic residues in halophilic adaptation was investigated by site-directed mutagenesis of malate dehydrogenase (MalDH) from Haloarcula marismortui. In all of 40 non-halophilic homologous proteins, the position aligned with E243 in halophilic MalDH is occupied by a non-acidic amino acid, most frequently by arginine. The E243R mutant of halophilic MalDH was constructed, over-expressed in Escherichia coli, renatured and purified. Its salt-dependent catalytic activity was not affected compared to the wild-type enzyme and both proteins have the same Km values for their substrates. The resistance to denaturation of the mutant was compared to that of the wild-type protein in different physiological salt (NaCl or KCl) and temperature conditions and interpreted in terms of classical quasi-thermodynamic parameters. The mutant is more halophilic than the wild-type protein; it is more sensitive to temperature and requires significantly higher concentrations of NaCl or KCl for equivalent stability. These results highlight the role of acidic amino acids in halophilic behaviour and are in agreement with a model in which these amino acids act cooperatively to organise hydrated ion binding to the protein.